Kang Zheng

BDNF Promotes Differentiation and Maturation of Adult-born Neurons through GABAergic Transmission

Brain-derived neurotrophic factor (BDNF) has been implicated in regulating adult neurogenesis in the subgranular zone (SGZ) of the dentate gyrus; however, the mechanism underlying this regulation remains unclear. In this study, we found that Bdnf mRNA localized to distal dendrites of dentate gyrus granule cells isolated from wild-type (WT) mice, but not from Bdnfklox/klox mice where the long 3′ untranslated region (UTR) of Bdnf mRNA is truncated. KCl-induced membrane depolarization stimulated release of dendritic BDNF translated from long 3′ UTR Bdnf mRNA in cultured hippocampal neurons, but not from short 3′ UTR Bdnf mRNA. Bdnfklox/klox mice exhibited reduced expression of glutamic acid decarboxylase 65 (a GABA synthase), increased proliferation of progenitor cells, and impaired differentiation and maturation of newborn neurons in the SGZ. These deficits in adult neurogenesis were rescued with administration of phenobarbital, an enhancer of GABAA receptor activity. Furthermore, we observed similar neurogenesis deficits in mice where the receptor for BDNF, TrkB, was selectively abolished in parvalbumin (PV)-expressing GABAergic interneurons. Thus, our data suggest that locally synthesized BDNF in dendrites of granule cells promotes differentiation and maturation of progenitor cells in the SGZ by enhancing GABA release, at least in part, from PV-expressing GABAergic interneurons.

TrkB signaling in parvalbumin-positive interneurons is critical for gamma-band network synchronization in hippocampus

Although brain-derived neurotrophic factor (BDNF) is known to regulate circuit development and synaptic plasticity, its exact role in neuronal network activity remains elusive. Using mutant mice (TrkB-PV−/−) in which the gene for the BDNF receptor, tyrosine kinase B receptor (trkB), has been specifically deleted in parvalbumin-expressing, fast-spiking GABAergic (PV+) interneurons, we show that TrkB is structurally and functionally important for the integrity of the hippocampal network. The amplitude of glutamatergic inputs to PV+ interneurons and the frequency of GABAergic inputs to excitatory pyramidal cells were reduced in the TrkB-PV−/− mice. Functionally, rhythmic network activity in the gamma-frequency band (30–80 Hz) was significantly decreased in hippocampal area CA1. This decrease was caused by a desynchronization and overall reduction in frequency of action potentials generated in PV+ interneurons of TrkB-PV−/− mice. Our results show that the integration of PV+ interneurons into the hippocampal microcircuit is impaired in TrkB-PV−/− mice, resulting in decreased rhythmic network activity in the gamma-frequency band.

Electrophysiological studies on galanin effects in brain – progress during the last six years

The effects of galanin and galanin fragments have been studied on neurons in various brain regions of rodents using electrophysiological techniques. Here, we mainly review reports published during the last six years, that is after the second galanin symposium in 1998. These papers deal with locus coeruleus (LC), the hippocampal formation (HF), hypothalamus, the nucleus of the diagonal band of Broca (DBB) and the dorsal vagal complex (DVC). In most cases galanin has an inhibitory effect by increasing a potassium conductance or reducing a calcium conductance. In LC, beside a direct inhibitory effect, galanin exerts an indirect effect enhancing the noradrenaline-induced hyperpolarization. In the HF, galanin (1-15), but not galanin (1-29), induces hyperpolarization in CA3 pyramidal neurons. Inhibitory effects of galanin on several forms of synaptic plasticity including long-term potentiation, frequency facilitation and paired-pulse facilitation have also been demonstrated in normal and transgenic animals. In the hypothalamic arcuate nucleus galanin has a presynaptic action inhibiting glutamate release, as well as a postsynaptic effect via the galanin R1 receptor. In the DVC, galanin inhibits dorsal vagal motor neurons projecting to the stomach by activation of a postsynaptic galanin receptor. However, excitatory effects of galanin have also been reported in several regions, such as the DBB nucleus, where galanin increases excitability by decreasing a K+ conductance. Taken together, electrophysiological studies have further supported the role of galanin as a neurotransmitter/neuromodulator in the brain.

Postendocytotic traffic of the galanin R1 receptor: a lysosomal signal motif on the cytoplasmic terminus.

The neuropeptide galanin R1 receptor (GalR1) was tagged at its C terminus with EGFP (GalR1–EGFP) to study receptor localization and trafficking. In PC12 and HEK293 cells, functional GalR1–EGFP was expressed on the plasma membrane and internalized into cytoplasmic vesicles after galanin stimulation. The internalization was blocked by 0.4 M sucrose and by silencing of clathrin with siRNA methodology. Internalized GalR1–EGFP and LysoTracker, a lysosomal marker, overlapped in intracellular vesicles after prolonged galanin stimulation. This colocalization was strongly reduced after site-directed mutagenesis of the motif YXXØ on the C terminus of GalR1 (where Ø is a bulky hydrophobic residue and X any amino acid). Taken together, these data suggest that GalR1 is internalized via the clathrin-dependent, endocytic pathway and then, to a large extent, delivered to lysosomes for degradation through the lysosome-targeting signal YXXØ.

Constitutive and ligand-induced internalization of EGFP-tagged galanin R2 and Rl receptors in PC12 cells.

In the present experiments trafficking of the galanin R1-(GALR1) and, in particular, the galanin R2 receptor (GALR2) was studied after fusion with enhanced green fluorescent protein (GALR1-EGFP and GALR2-EGFP) and transfection into PC12 cells. Both fusion proteins were predominantly localized on the plasma membrane and internalized in a dose dependent manner after incubation with galanin. Preincubation with M35 or M40 did not prevent galanin-induced internalization of GALR1-EGFP or GALR2-EGFP. However, AR-M1896, a selective GALR2 agonist, caused GALR2, but not GALR1 internalization. Hyperosmotic sucrose inhibited internalization of GALR2-EGFP. After co-incubation with galanin, GALR2-EGFP was co-localized with internalized Texas Red transferrin, a marker of the clathrin endocytic pathway. Experiments with protein synthesis inhibition and Texas Red transferrin suggest that GALR2 is constitutively internalized. Studies in progress will show if this is the case also for GALR1.

Age-related impairments of synaptic plasticity in the lateral perforant path input to the dentate gyrus of galanin overexpressing mice

In the present study, electrophysiological recordings were made from hippocampal slices obtained from mice overexpressing galanin under the promoter for the platelet-derived growth factor-B (GalOE mice). In these mice, a particularly strong galanin expression is seen in the granule cell layer/mossy fibers. Paired-pulse facilitation (PPF) of excitatory postsynaptic field potentials (fEPSPs) at the lateral perforant path (LPP)-dentate gyrus synapses was elicited in the dentate gyrus after stimulation with different interpulse intervals. Slices from young adult wild-type (WT) animals showed significant PPF of the 2nd EPSP evoked with paired-pulse stimuli, while PPF was reduced in slices from young adult GalOE mice, as well as aged WT mice, but were not observed at all in slices from aged GalOE animals. Application of the putative galanin antagonist M35 increased PPF in slices from aged WT mice as well as from adult and aged GalOE mice, but had no effect in slices taken from young adult WT mice. These data indicate that galanin is involved in hippocampal synaptic plasticity, in particular in age-related reduction of synaptic plasticity in the LPP input to the dentate gyrus. Galaninergic mechanisms may therefore represent therapeutic targets for treatment of age-related memory deficits and Alzheimers disease.

GABAergic Terminals Are a Source of Galanin to Modulate Cholinergic Neuron Development in the Neonatal Forebrain

The distribution and (patho-)physiological role of neuropeptides in the adult and aging brain have been extensively studied. Galanin is an inhibitory neuropeptide that can coexist with γ-aminobutyric acid (GABA) in the adult forebrain. However, galanins expression sites, mode of signaling, impact on neuronal morphology, and colocalization with amino acid neurotransmitters during brain development are less well understood. Here, we show that galaninergic innervation of cholinergic projection neurons, which preferentially express galanin receptor 2 (GalR2) in the neonatal mouse basal forebrain, develops by birth. Nerve growth factor (NGF), known to modulate cholinergic morphogenesis, increases GalR2 expression. GalR2 antagonism (M871) in neonates reduces the in vivo expression and axonal targeting of the vesicular acetylcholine transporter (VAChT), indispensable for cholinergic neurotransmission. During cholinergic neuritogenesis in vitro, GalR2 can recruit Rho-family GTPases to induce the extension of a VAChT-containing primary neurite, the prospective axon. In doing so, GalR2 signaling dose-dependently modulates directional filopodial growth and antagonizes NGF-induced growth cone differentiation. Galanin accumulates in GABA-containing nerve terminals in the neonatal basal forebrain, suggesting its contribution to activity-driven cholinergic development during the perinatal period. Overall, our data define the cellular specificity and molecular complexity of galanin action in the developing basal forebrain.