Yuri Zilberter

Pyramidal cell communication within local networks in layer 2/3 of rat neocortex

The extent to which neocortical pyramidal cells function as a local network is determined by the strength and probability of their connections. By mapping connections between pyramidal cells we show here that in a local network of about 600 pyramidal cells located within a cylindrical volume of 200 μm × 200 μm of neocortical layer 2/3, an individual pyramidal cell receives synaptic inputs from about 30 other pyramidal neurons, with the majority of EPSP amplitudes in the 0.2–1.0 mV range. The probability of connection decreased from 0.09 to 0.01 with intercell distance (over the range 25–200 μm). Within the same volume, local interneuron (fast‐spiking non‐accomodating interneuron, FS)‐pyramidal cell connections were about 10 times more numerous, with the majority of connections being reciprocal. The probability of excitatory and inhibitory connections between pyramidal cells and FS interneurons decreased only slightly with distance, being in the range 0.5–0.75. Pyramidal cells in the local network received strong synaptic input during stimulation of afferent fibres in layers 1 and 6. Minimal‐like stimulation of layer 1 or layer 6 inputs simultaneously induced postsynaptic potentials in connected pyramidal cells as well as in pyramidal‐FS cell pairs. These inputs readily induced firing of pyramidal cells, although synaptically connected cells displayed different firing patterns. Unitary EPSPs in pyramidal‐pyramidal cell pairs did not detectably alter cell firing. FS interneurons fire simultaneously with pyramidal cells. In pyramidal‐FS cell pairs, both unitary EPSPs and IPSPs efficiently modulated cell firing patterns. We suggest that computation in the local network may proceed not only by direct pyramidal‐pyramidal cell communication but also via local interneurons. With such a high degree of connectivity with surrounding pyramidal cells, local interneurons are ideally poised to both coordinate and expand the local pyramidal cell network via pyramidal‐ interneuron‐ pyramidal communication.

Amyloid β-Induced Neuronal Hyperexcitability Triggers Progressive Epilepsy

Alzheimers disease is associated with an increased risk of unprovoked seizures. However, the underlying mechanisms of seizure induction remain elusive. Here, we performed video-EEG recordings in mice carrying mutant human APPswe and PS1dE9 genes (APdE9 mice) and their wild-type littermates to determine the prevalence of unprovoked seizures. In two recording episodes at the onset of amyloid β (Aβ) pathogenesis (3 and 4.5 months of age), at least one unprovoked seizure was detected in 65% of APdE9 mice, of which 46% had multiple seizures and 38% had a generalized seizure. None of the wild-type mice had seizures. In a subset of APdE9 mice, seizure phenotype was associated with a loss of calbindin-D28k immunoreactivity in dentate granular cells and ectopic expression of neuropeptide Y in mossy fibers. In APdE9 mice, persistently decreased resting membrane potential in neocortical layer 2/3 pyramidal cells and dentate granule cells underpinned increased network excitability as identified by patch-clamp electrophysiology. At stimulus strengths evoking single-component EPSPs in wild-type littermates, APdE9 mice exhibited decreased action potential threshold and burst firing of pyramidal cells. Bath application (1 h) of Aβ1–42 or Aβ25–35 (proto-)fibrils but not oligomers induced significant membrane depolarization of pyramidal cells and increased the activity of excitatory cell populations as measured by extracellular field recordings in the juvenile rodent brain, confirming the pathogenic significance of bath-applied Aβ (proto-)fibrils. Overall, these data identify fibrillar Aβ as a pathogenic entity powerfully altering neuronal membrane properties such that hyperexcitability of pyramidal cells culminates in epileptiform activity.

Excitatory GABA in Rodent Developing Neocortex In Vitro

GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential (Em), reversal potential of GABA (E(GABA)), and threshold of action potential generation (Vthr). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-d-aspartate and GABA channel recordings in rodent brain slices to measure both Em and E(GABA) in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2-P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized Vthr. The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5-P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl(-)]i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

Endocannabinoid-independent retrograde signaling at inhibitory synapses in layer 2/3 of neocortex: involvement of vesicular glutamate transporter 3.

Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex. Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laser-scanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 μm) or intracellular Cl- concentrations (4-145 mm). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.

GABA action in immature neocortical neurons directly depends on the availability of ketone bodies

In the early postnatal period, energy metabolism in the suckling rodent brain relies to a large extent on metabolic pathways alternate to glucose such as the utilization of ketone bodies (KBs). However, how KBs affect neuronal excitability is not known. Using recordings of single NMDA and GABA‐activated channels in neocortical pyramidal cells we studied the effects of KBs on the resting membrane potential (Em) and reversal potential of GABA‐induced anionic currents (EGABA), respectively. We show that during postnatal development (P3–P19) if neocortical brain slices are adequately supplied with KBs, Em and EGABA are both maintained at negative levels of about −83 and −80 mV, respectively. Conversely, a KB deficiency causes a significant depolarization of both Em (>5 mV) and EGABA (>15 mV). The KB‐mediated shift in EGABA is largely determined by the interaction of the NKCC1 cotransporter and Cl−/HCO3 transporter(s). Therefore, by inducing a hyperpolarizing shift in Em and modulating GABA signaling mode, KBs can efficiently control the excitability of neonatal cortical neurons.

Postnatal changes in somatic γ‐aminobutyric acid signalling in the rat hippocampus

During postnatal development of the rat hippocampus, γ‐aminobutyric acid (GABA) switches its action on CA3 pyramidal cells from excitatory to inhibitory. To characterize the underlying changes in the GABA reversal potential, we used somatic cell‐attached recordings of GABA(A) and N‐methyl‐d‐aspartate channels to monitor the GABA driving force and resting membrane potential, respectively. We found that the GABA driving force is strongly depolarizing during the first postnatal week. The strength of this depolarization rapidly declines with age, although GABA remains slightly depolarizing, by a few millivolts, even in adult neurons. Reduction in the depolarizing GABA driving force was due to a progressive negative shift of the reversal potential of GABA currents. Similar postnatal changes in GABA signalling were also observed using the superfused hippocampus preparation in vivo, and in the hippocampal interneurons in vitro. We also found that in adult pyramidal cells, somatic GABA reversal potential is maintained at a slightly depolarizing level by bicarbonate conductance, chloride‐extrusion and chloride‐loading systems. Thus, the postnatal excitatory‐to‐inhibitory switch in somatic GABA signalling is associated with a negative shift of the GABA reversal potential but without a hyperpolarizing switch in the polarity of GABA responses. These results also suggest that in adult CA3 pyramidal cells, somatic GABAergic inhibition takes place essentially through shunting rather than hyperpolarization. Apparent hyperpolarizing GABA responses previously reported in the soma of CA3 pyramidal cells are probably due to cell depolarization during intracellular or whole‐cell recordings.

Brain-derived neurotrophic factor controls functional differentiation and microcircuit formation of selectively isolated fast-spiking GABAergic interneurons

GABAergic interneurons with high‐frequency firing, fast‐spiking (FS) cells, form synapses on perisomatic regions of principal cells in the neocortex and hippocampus to control the excitability of cortical networks. Brain‐derived neurotrophic factor (BDNF) is essential for the differentiation of multiple interneuron subtypes and the formation of their synaptic contacts. Here, we examined whether BDNF, alone or in conjunction with sustained KCl‐induced depolarization, drives functional FS cell differentiation and the formation of inhibitory microcircuits. Homogeneous FS cell cultures were established by target‐specific isolation using the voltage‐gated potassium channel 3.1b subunit as the selection marker. Isolated FS cells expressed parvalbumin, were surrounded by perineuronal nets, formed immature inhibitory connections and generated slow action potentials at 12 days in vitro. Brain‐derived neurotrophic factor (BDNF) promoted FS cell differentiation by increasing the somatic diameter, dendritic branching and the frequency of action potential firing. In addition, BDNF treatment led to a significant up‐regulation of synaptophysin and vesicular GABA transporter expression, components of the synaptic machinery critical for GABA release, which was paralleled by an increase in synaptic strength. Long‐term membrane depolarization alone was detrimental to dendritic branching. However, we observed that BDNF and KCl exerted additive effects, as reflected by the significantly accelerated maturation of synaptic contacts and high discharge frequencies, and was required for the formation of reciprocal connections between FS cells. Our results show that BDNF, along with membrane depolarization, is critical for FS cells to establish inhibitory circuitries during corticogenesis.

Energy substrate availability as a determinant of neuronal resting potential, GABA signaling and spontaneous network activity in the neonatal cortex in vitro

J. Neurochem. (2009) 112, 900–912.

Non-fibrillar β-amyloid abates spike-timing-dependent synaptic potentiation at excitatory synapses in layer 2/3 of the neocortex by targeting postsynaptic AMPA receptors

Cognitive decline in Alzheimers disease (AD) stems from the progressive dysfunction of synaptic connections within cortical neuronal microcircuits. Recently, soluble amyloid β protein oligomers (Aβols) have been identified as critical triggers for early synaptic disorganization. However, it remains unknown whether a deficit of Hebbian‐related synaptic plasticity occurs during the early phase of AD. Therefore, we studied whether age‐dependent Aβ accumulation affects the induction of spike‐timing‐dependent synaptic potentiation at excitatory synapses on neocortical layer 2/3 (L2/3) pyramidal cells in the APPswe/PS1dE9 transgenic mouse model of AD. Synaptic potentiation at excitatory synapses onto L2/3 pyramidal cells was significantly reduced at the onset of Aβ pathology and was virtually absent in mice with advanced Aβ burden. A decreased α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)/N‐methyl‐d‐aspartate (NMDA) receptor‐mediated current ratio implicated postsynaptic mechanisms underlying Aβ synaptotoxicity. The integral role of Aβols in these processes was verified by showing that pretreatment of cortical slices with Aβ(25−35)ols disrupted spike‐timing‐dependent synaptic potentiation at unitary connections between L2/3 pyramidal cells, and reduced the amplitude of miniature excitatory postsynaptic currents therein. A robust decrement of AMPA, but not NMDA, receptor‐mediated currents in nucleated patches from L2/3 pyramidal cells confirmed that Aβols perturb basal glutamatergic synaptic transmission by affecting postsynaptic AMPA receptors. Inhibition of AMPA receptor desensitization by cyclothiazide significantly increased the amplitude of excitatory postsynaptic potentials evoked by afferent stimulation, and rescued synaptic plasticity even in mice with pronounced Aβ pathology. We propose that soluble Aβols trigger the diminution of synaptic plasticity in neocortical pyramidal cell networks during early stages of AD pathogenesis by preferentially targeting postsynaptic AMPA receptors.

Complementary distribution of type 1 cannabinoid receptors and vesicular glutamate transporter 3 in basal forebrain suggests input‐specific retrograde signalling by cholinergic neurons

Basal forebrain cholinergic neurons project to diverse cortical and hippocampal areas and receive reciprocal projections therefrom. Maintenance of a fine‐tuned synaptic communication between pre‐ and postsynaptic cells in neuronal circuitries also requires feedback mechanisms to control the probability of neurotransmitter release from the presynaptic terminal. Release of endocannabinoids or glutamate from a postsynaptic neuron has been identified as a means of retrograde synaptic signalling. Presynaptic action of endocannabinoids is largely mediated by type 1 cannabinoid (CB1) receptors, while fatty‐acid amide hydrolase (FAAH) is involved in inactivating some endocannabinoids postsynaptically. Alternatively, vesicular glutamate transporter 3 (VGLUT3) controls release of glutamate from postsynaptic cells. Here, we studied the distribution of CB1 receptors, FAAH and VGLUT3 in cholinergic basal forebrain nuclei of mouse and rat. Cholinergic neurons were devoid of CB1 receptor immunoreactivity. A fine CB1 receptor‐immunoreactive (ir) fibre meshwork was present in medial septum, diagonal bands and nucleus basalis. In contrast, the ventral pallidum and substantia innominata received dense CB1 receptor‐ir innervation and cholinergic neurons received CB1 receptor‐ir presumed synaptic contacts. Consistent with CB1 receptor distribution, FAAH‐ir somata were abundant in basal forebrain and appeared in contact with CB1 receptor‐containing terminals. Virtually all cholinergic neurons were immunoreactive for FAAH. A significant proportion of cholinergic cells exhibited VGLUT3 immunoreactivity in medial septum, diagonal bands and nucleus basalis, and were in close apposition to VGLUT3‐ir terminals. VGLUT3 immunoreactivity was largely absent in ventral pallidum and substantia innominata. We propose that specific subsets of cholinergic neurons may utilize endocannabinoids or glutamate for retrograde control of the efficacy of input synapses, and the mutually exclusive complementary distribution pattern of CB1 receptor‐ir and VGLUT3‐ir fibres in basal forebrain suggests segregated input‐specific signalling mechanisms by cholinergic neurons.