Tibor Harkany

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.

Increased abundance of opioid receptor heteromers after chronic morphine administration.

The μ-δ opioid heteromer may be a target for alleviation of chronic pain. Drug-Induced Heteromers Opioid receptors are G protein–coupled receptors and are divided into μ, δ, and κ subtypes. Homomers of μ or δ receptors signal through a Gαi-mediated pathway; however, these receptor subtypes can also form heteromers that signal through Gαz- or β-arrestin2–mediated pathways. Although morphine analgesia is mediated primarily through the μ receptor, δ receptor ligands can potentiate μ receptor–mediated signaling, suggesting that the μ-δ heteromer may also be involved in morphine analgesia. Gupta et al. developed an antibody that selectively recognizes μ-δ heteromers and found that the abundance of the μ-δ heteromer in mice increased with chronic administration of morphine. These increases were detected in regions of the brain that are involved in the modulation of pain transmission. These results suggest that the μ-δ heteromer may be a candidate for therapies to alleviate chronic pain syndromes. The μ and δ types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the μ-δ heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of μ-δ heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the μ-δ heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

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 −80u2003mV, respectively. Conversely, a KB deficiency causes a significant depolarization of both Em (>5u2003mV) and EGABA (>15u2003mV). 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.

System A Transporter SAT2 Mediates Replenishment of Dendritic Glutamate Pools Controlling Retrograde Signaling by Glutamate

Glutamate mediates several modes of neurotransmission in the central nervous system including recently discovered retrograde signaling from neuronal dendrites. We have previously identified the system N transporter SN1 as being responsible for glutamine efflux from astroglia and proposed a system A transporter (SAT) in subsequent transport of glutamine into neurons for neurotransmitter regeneration. Here, we demonstrate that SAT2 expression is primarily confined to glutamatergic neurons in many brain regions with SAT2 being predominantly targeted to the somatodendritic compartments in these neurons. SAT2 containing dendrites accumulate high levels of glutamine. Upon electrical stimulation in vivo and depolarization in vitro, glutamine is readily converted to glutamate in activated dendritic subsegments, suggesting that glutamine sustains release of the excitatory neurotransmitter via exocytosis from dendrites. The system A inhibitor MeAIB (alpha-methylamino-iso-butyric acid) reduces neuronal uptake of glutamine with concomitant reduction in intracellular glutamate concentrations, indicating that SAT2-mediated glutamine uptake can be a prerequisite for the formation of glutamate. Furthermore, MeAIB inhibited retrograde signaling from pyramidal cells in layer 2/3 of the neocortex by suppressing inhibitory inputs from fast-spiking interneurons. In summary, we demonstrate that SAT2 maintains a key metabolic glutamine/glutamate balance underpinning retrograde signaling by dendritic release of the neurotransmitter glutamate.

Cracking Down on Inhibition: Selective Removal of GABAergic Interneurons from Hippocampal Networks

Inhibitory (GABAergic) interneurons entrain assemblies of excitatory principal neurons to orchestrate information processing in the hippocampus. Disrupting the dynamic recruitment as well as the temporally precise activity of interneurons in hippocampal circuitries can manifest in epileptiform seizures, and impact specific behavioral traits. Despite the importance of GABAergic interneurons during information encoding in the brain, experimental tools to selectively manipulate GABAergic neurotransmission are limited. Here, we report the selective elimination of GABAergic interneurons by a ribosome inactivation approach through delivery of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro as well as in the mouse and rat hippocampus in vivo. We demonstrate the selective loss of GABAergic—but not glutamatergic—synapses, reduced GABA release, and a shift in excitation/inhibition balance in mixed cultures of hippocampal neurons exposed to SAVAs. We also show the focal and indiscriminate loss of calbindin+, calretinin+, parvalbumin/system A transporter 1+, somatostatin+, vesicular glutamate transporter 3 (VGLUT3)/cholecystokinin/CB1 cannabinoid receptor+ and neuropeptide Y+ local-circuit interneurons upon SAVA microlesions to the CA1 subfield of the rodent hippocampus, with interneuron debris phagocytosed by infiltrating microglia. SAVA microlesions did not affect VGLUT1+ excitatory afferents. Yet SAVA-induced rearrangement of the hippocampal circuitry triggered network hyperexcitability associated with the progressive loss of CA1 pyramidal cells and the dispersion of dentate granule cells. Overall, our data identify SAVAs as an effective tool to eliminate GABAergic neurons from neuronal circuits underpinning high-order behaviors and cognition, and whose manipulation can recapitulate pathogenic cascades of epilepsy and other neuropsychiatric illnesses.

Diacylglycerol lipase α manipulation reveals developmental roles for intercellular endocannabinoid signaling

Endocannabinoids are small signaling lipids, with 2-arachidonoylglycerol (2-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic 2-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing 2-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB1 cannabinoid receptors (CB1Rs) remains poorly understood. Here, we first show that cholinergic innervation of CA1 pyramidal cells of the hippocampus is sensitive to the genetic disruption of 2-AG signaling in DAGLα null mice. Next, we exploit a hybrid COS-7-cholinergic neuron co-culture system to demonstrate that heterologous DAGLα overexpression spherically excludes cholinergic growth cones from 2-AG-rich extracellular environments, and minimizes cell-cell contact in vitro. CB1R-mediated exclusion responses lasted 3 days, indicating sustained spherical 2-AG availability. Overall, these data suggest that extracellular 2-AG concentrations can be sufficient to activate CB1Rs along discrete spherical boundaries to modulate neuronal responsiveness.

In vivo labeling of rabbit cholinergic basal forebrain neurons with fluorochromated antibodies

Cholinergic basal forebrain neurons (CBFN) expressing the low-affinity neurotrophin receptor p75 (p75NTR) were previously selectively labeled in vivo with carbocyanine 3 (Cy3)-tagged anti-p75NTR, but the applied 192IgG-conjugates recognized p75NTR only in rat. The antibody ME 20.4 raised against human p75NTR had been shown to cross-react with the receptor in monkey, raccoon, sheep, cat, dog, pig and rabbit. Hence, for in vivo labeling of rabbit CBFN in the present study, ME 20.4 was fluorochromated with Cy3-N-hydroxysuccinimide ester and purified Cy3-ME 20.4 was injected intracerebroventricularly. Two days post-injection, clusters of Cy3-ME 20.4 were found in CBFN displaying choline acetyltrans-ferase-immunoreactivity. Following photoconversion, electron microscopy revealed fluorochromated antibodies in secondary lysosomes. In conclusion, Cy3-ME 20.4 might become an appropriate marker for CBFN in live and fixed tissues of various mammalian species.

CB1 Cannabinoid Receptors: Molecular Biology, Second Messenger Coupling and Polarized Trafficking in Neurons

The type 1 cannabinoid receptor (CB 1 receptor) is considered to be the most abundant G protein-coupled receptor (GPCR) in the mammalian brain. The presence and highly compartmentalized cellular distribution of CB 1 receptors in neurons localized to corticolimbic areas, basal ganglia, cerebellum, and brain- stem accounts for the majority of behavioral actions associated with cannabinoid drugs. The discovery of endocannabinoids led to an avalanche of data showing that signaling at this GPCR is critical for, e.g., neurogenesis, neural development, synaptic plasticity, learning and memory, food intake, and energy metabolism. In contrast, deficient CB 1 receptor expression or coupling to downstream signal transduction cascades contributes to the neuropathogenesis of a broad variety of neurological and metabolic disorders with selective pharmacological modula- tion of CB 1 receptor availability and activity being a prime target for therapeutic intervention. Here, we summarize contemporary knowledge on the regulation of CB 1 receptor expression in the central nervous system and describe the context- dependent recruitment of second messengers to this receptor. Finally, we present the concept that CB 1 receptor bioavailability together with its momentary sign- aling activity on neuronal membranes defines the efficacy of endocannabinoid signaling such that a fine-tuned control of synaptic efficacy and plasticity may be achieved.

Rabbit Forebrain cholinergic system

Although the rabbit brain, in particular the basal forebrain cholinergic system, has become a common model for neuropathological changes associated with Alzheimers disease, detailed neuroanatomical studies on the morphological organization of basal forebrain cholinergic nuclei and on their output pathways are still awaited. Therefore, we performed quantitative choline acetyltransferase (ChAT) immunocytochemistry to localize major cholinergic nuclei and to determine the number of respective cholinergic neurons in the rabbit forebrain. The density of ChAT‐immunoreactive terminals in layer V of distinct neocortical territories and in hippocampal subfields was also measured. Another cholinergic marker, the low‐affinity neurotrophin receptor (p75NTR), was also employed to identify subsets of cholinergic neurons. Double‐immunofluorescence labeling of ChAT and p75NTR, calbindin D‐28k (CB), parvalbumin, calretinin, neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase, or substance P was used to elucidate the neuroanatomical borders of cholinergic nuclei and to analyze the neurochemical complexity of cholinergic cell populations. Cholinergic projection neurons with heterogeneous densities were found in the medial septum, vertical and horizontal diagonal bands of Broca, ventral pallidum, and magnocellular nucleus basalis (MBN)/substantia innominata (SI) complex; cholinergic interneurons were observed in the caudate nucleus, putamen, accumbens nucleus, and olfactory tubercule, whereas the globus pallidus was devoid of cholinergic nerve cells. Cholinergic interneurons were frequently present in the hippocampus and to a lesser extent in cerebral cortex. Cholinergic projection neurons, except those localized in SI, abundantly expressed p75NTR, and a subset of cholinergic neurons in posterior MBN was immunoreactive for CB and nNOS. A strict laminar distribution pattern of cholinergic terminals was recorded both in the cerebral cortex and in CA1–CA3 and dentate gyrus of the hippocampus. In summary, the structural organization and chemoarchitecture of rabbit basal forebrain may be considered as a transition between that of rodents and that of primates. J. Comp. Neurol. 460:597–611, 2003.

Molecular mechanisms of neuronal specification

What are the precise molecular and cellular mechanisms that the human brain exploits to encode consciousness, identity and thought? This undoubtedly remains one of the greatest scientific challenges facing mankind. Unravelling the structural and functional diversity of neurons, an estimated 10 of which populate the cerebral cortex alone (Rakic, 2009), and explaining the molecular principles that integrate these cells into coherent and dynamic networks have been at the forefront of neuroscience research for the past century. Key inferences of our understanding include the following. Firstly, that each prospective neuron harbours the transcriptional code that defines its placement and basic structural and functional make-up (Fishell & Hanashima, 2008). Secondly, the computational power of the human brain is entrained by synapses, specialized junctions of communication. The placement of synapses is precisely defined during the growth of the axon (Song & Poo, 1999) that emerges from developing neurons to function as a conduit of electrical impulses representing packets of information. Thirdly, the continuous and ‘on-demand’ adaptation of synapses provides the substrate for learning and memory. Finally, excess pruning of synaptic contacts impairs the brain’s capacity to recruit an essential minimum of neurons into computational networks (Kantor & Kolodkin, 2003). This has the effect of preventing the execution of vital commands. The present Special Issue entitled ‘Molecular Mechanisms of Neuronal Specification’ encompasses 17 reviews and original research articles addressing these rules with eminent precision and attention to detail (Fig. 1). The emergence of primary ‘hot-spots’ derived from the converging interest of some of the leading neurodevelopmental research groups in this Special Issue is naturally exciting, and it is hoped that they will serve as guideposts for innovative studies for the years to come. This ensemble thematically opens by surveying the molecular regulation of the earliest events of nervous system development, when the neural and border domains of the embryonic ectoderm define the central and peripheral nervous systems respectively, and by outlining a novel model of how extracellular signals interact to coordinate the specification and regionalization of border cells (Patthey & Gunhaga, 2011). Our attention then turns towards a series of original research and review articles providing a comprehensive account of the cellautonomous and intercellular mechanisms to address how, where and when neuronal diversity is generated. Some of these studies focus on the formation of the cerebral cortex (Anastasiades &Butt, 2011; Antypa et al., 2011), whose uniquely ordered (laminated) cellular organization and functional precision to orchestrate specific behaviours relies on the timely migration and synaptic wiring of interneurons and pyramidal cells, and has fascinated many generations of neurobiologists. Molecular principles of GABAergic interneurons come to the fore as the unprecedented variety of these local-circuit components of cortical networks emphasizes that overarching cascades of instructive mechanisms must continuously operate to facilitate the attaining of neuronal identity (Anastasiades & Butt, 2011). Three additional articles broaden the concept of the transcription factor requirements of neuronal differentiation into a general rule by showing central roles for Shox2 in the specification of discriminative touch-sensitive neurons in sensory dorsal root ganglia (Abdo et al., 2011), and transcriptional cascades converging on Lmx1b ⁄ Pet1 and giving rise to hindbrain serotonergic neurons (Kiyasova & Gaspar, 2011). A bone morphogenetic protein-induced network of transcription factors, with central roles for Ascl1 and Phox2b, with sequential expression is described as inducing autonomic neuron differentiation in sympathetic ganglia (Rohrer, 2011). A unifying theme of these articles is the fascinating concept that the expression of transcription factors, and their combinations, is fate instructive, required to prospectively determine the intrinsic molecular and electrical makeup of differentiated neurons, and their maintenance until adulthood is critical for the continued manifestation of neuronal subtype characteristics, as well as neuronal survival. Intriguingly, Kiyasova & Gaspar (2011) put forward the hypothesis that neurons may rely on the very same transcription factors that instruct their identity (e.g. Pet1) to orchestrate axonal growth and guidance, and the formation of hierarchical synaptic connectivity maps. Nevertheless, unwavering efforts are directed to discover novel signalling networks that control neuronal migration (Antypa et al., 2011; Manent et al., 2011) and ⁄ or axonal growth and guidance (Chenaux & Henkemeyer, 2011;Oudin et al., 2011). The impact of furthering our understanding of molecular signalling events through the genetic dissection of bidirectional ligand–receptor interactions between the axon and its target is highlighted by Chenaux & Henkemeyer (2011), whose work closes in on the EphB–ephrin-B interaction during axonal pathfinding of retinal ganglion cells. It is one thing to understand how a single axon navigates towards its target, but understanding the ordered growth of many axons to establish large-scale topologically precise sensory maps is entirely another. Here, Wu et al. (2011) discuss the myriad of diffusible axon guidance cues that orchestrate the precisely timed and topographically correct innervation of target cells during the formation of the whiskerto-barrel (somatosensory) cortex circuitry. In turn, Imai & Sakano (2011) propose the appealing model that the establishment of precise topographic maps in sensory systems is through the direct interplay and ‘self-organization’ of the growing afferent axons. Is there a specific spatial resolution at which information must be gained to appreciate the range of signalling mechanisms in vivo? Antypa et al. (2011) have succeeded in exploiting the strength of high-throughput array technologies to elucidate the spatial constraints of instructive signals in relation to discrete subcontingents of Correspondence: Tibor Harkany, European Neuroscience Institute, as above. E-mail: t.harkany@abdn.ac.uk