Specific gene expression in unmyelinated dorsal root ganglion neurons in nonhuman primates by intra-nerve injection of AAV 6 vector

霊長類においてウイルスベクターを用いた痛覚神経への遺伝子導入に成功 --神経障害性疼痛治療への応用に期待--. 京都大学プレスリリース. 2021-08-12.Adeno-associated virus 6 has been proposed as a potential vector candidate for specific gene expression in pain-related dorsal root ganglion neurons, but this has not been confirmed in nonhuman primates. The aim of our study was to analyze the transduction efficiency and target specificity of this viral vector in the common marmoset by comparing with those in the rat. When green fluorescent protein-expressing serotype-6 vector was injected into the sciatic nerve, the efficiency of gene expression in dorsal root ganglion neurons was comparable in both species. We found that the serotype-6 vector was largely specific to the pain-related ganglion neurons in the marmoset as well as in the rat, whereas the serotype-9 vector resulted in contrasting effects in the two species. Neither AAV6 nor AAV9 resulted in DRG toxicity when administered via the sciatic nerve, suggesting this as a safer route of sensory nerve transduction than the currently used intrathecal or intravenous administrative routes. Furthermore, the adeno-associated virus 6 vector could be an optimal serotype for gene therapy for human chronic pain that has minimal effect on other somatosensory functions of dorsal root ganglion neurons

Cerebellar Globular Cells Receive Monoaminergic Excitation and Monosynaptic Inhibition from Purkinje Cells

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control

IPSCs recorded from three types of small inhibitory interneurons underneath the Purkinje cell layer.

<p>(<i>A</i>) Representative sIPSCs. (left) a small Golgi cell (s-GoC), (middle) a small fusiform Lugaro cell (sf-LC), (right) a globular cell (GlC). Neurons were voltage-clamped at −70 mV in the presence of 1 mM kynurenic acid (or 10 µM NBQX and 30 µM APV). (<i>B</i>) Averaged frequency and amplitude of sIPSCs recorded from the three types of small interneurons. (<i>C</i>) Traces of sIPSCs in a GlC. Horizontal bar indicates perfusion of a GABA_A receptor antagonist, SR95531. (<i>D</i>) Miniature IPSCs recorded in a slow sweep (left half). Fast sweep traces of mIPSCs (gray) are superimposed (right half), and thick lines show averaged traces. These current traces were observed in an s-GoC, sf-LC, and GlC, as indicated. (<i>E</i>) Histogram for comparing averaged frequencies and amplitudes of mIPSCs. (<i>F</i>) Histogram showing frequencies of occurrence of TTX-sensitive sIPSC. (<i>G</i> and <i>H</i>) Rise and decay phases of mIPSC traces were fitted by a single exponential function, respectively. Pooled mean values for the rise (<i>G</i>) and decay (<i>H</i>) time constants of mIPSCs in s-GoCs (<i>n</i> = 7), sf-LCs (<i>n</i> = 4), and GlCs (<i>n</i> = 7) are shown. *** P<0.001, one-way ANOVA with Tukey's post test.</p

EPSCs recorded from cerebellar globular cells.

<p>Globular cells were voltage-clamped at −70 mV in the presence of 20 µM SR95531 and 1 µM strychnine. (<i>A</i>) (upper traces) example of sEPSCs recorded from a globular cell. (lower traces) a nonselective ionotropic glutamate receptor antagonist, kynurenic acid, completely blocked sEPSCs. (<i>B</i>) Individual sEPSCs (20 gray traces) were aligned at their rise time and superimposed. The averaged trace is shown by a black curve. (<i>C</i>) EPSCs evoked by double shock granular layer stimulation with changing intervals (10, 20, 40, and 80 ms) are superposed. (<i>D</i>) Ratio of the second responses to the first ones is plotted as a function of the interpulse interval (<i>n</i> = 3−5).</p

IPSCs in globular cells evoked by extracellular stimulation.

<p>(<i>A</i>) Paired pulse facilitation of IPSCs evoked in globular cells. Raw traces (gray, <i>n</i> = 12) and an averaged trace (black) of the IPSCs. (<i>B</i>) The paired-pulse facilitation of IPSCs in globular cells is plotted as a function of the interpulse interval (<i>n</i> = 3−7). (<i>C</i>) Frequency-dependent changes of evoked IPSCs. Twelve sweeps were averaged for each trace. The time scale bar indicates 200 ms (upper), 50 ms (middle), and 30 ms (lower). Stimulus artifact traces were eliminated at the basal current level. (<i>D</i>) The mean amplitudes of phasic IPSCs were measured relative to the current level preceding each stimulus artifact and normalized to the first IPSC amplitude of each train (50 Hz, <i>n</i> = 5; 100 Hz, <i>n</i> = 4). (<i>E</i>) The mean amplitudes of the basal current shift were measured immediately preceding each stimulus artifact.</p

Inhibitory synaptic connections between Purkinje cells and globular cells, and a globular cell-incorporated microcircuit.

<p>(<i>A</i>) Arrangement for paired recordings from a globular cell (GlC) and a Purkinje cell (PC) (PC1 or PC2). (<i>B</i>) Simple spike discharges of PC1 (lower) and sIPSCs in the GlC (upper). Action potentials indicated by red arrowheads caused IPSCs within a 2-ms delay following each action potential. (<i>C</i>) Distributions of time lags between peaks of action potentials in PC1 and onset of IPSCs in the GlC of (<i>B</i>). (<i>D</i>) Cross-correlogram of times of spike-peak and of sIPSC-onset recorded from the same pair in (<i>B</i>). (<i>E</i>) Superimposed traces of the action potentials in the presynaptic PC1 (upper) and individual IPSCs in the GlC (lower). Twenty traces were aligned with respect to the time course of the onset of the presynaptic action potentials. Six spikes failed to evoke IPSCs. (<i>F</i>) A few action potentials of PC2 (lower) caused sIPSCs in the GlC (upper), as shown by red arrowheads. (<i>G</i>) Distributions of time lags obtained in paired recordings from PC2 and the GlC of (<i>F</i>). (<i>H</i>) Cross-correlogram of times obtained from paired recordings in (<i>F</i>). (<i>I</i>) Paired whole-cell recordings from a PC and a GlC. Depolarizing stimulation (−65 to +10 mV, 1-ms duration) was applied to the presynaptic PC with a 2-sec interval. Superimposed fifty traces of presynaptic whole cell currents in the PC (upper) and IPSCs in the GlC (lower) are shown, respectively. (<i>J</i>) The amplitude histogram was obtained from 100 evoked IPSCs recorded in the same pair of (<i>I</i>). (<i>K</i>) A schematic representation of the microcircuit with GlC predicted by the previous anatomical <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029663#pone.0029663-Simat1" target="_blank">[6]</a> and present studies: the PC-GlC functional connection (thick line) is indicated in the local circuit of cerebellar cortex. BC: basket cell, SC: stellate cell, GoC: Golgi cell, GrC: granule cell.</p

Firing properties of inhibitory interneurons located close to the Purkinje cell layer.

<p>(<i>A–D</i>), Current-clamp recordings were performed to examine firing in response to an injection current step (+100 pA, 400 ms-duration, bottom) of small Golgi cell (s-GoC, <i>A</i>), small fusiform Lugaro cell (sf-LC, <i>B</i>), globular cell (GlC, <i>C</i>), and basket cell (BC, <i>D</i>). Dot lines indicate −55 mV. (<i>E</i>) Mean firing frequencies in response to injected currents of different amplitudes in s-GoCs (black circles, <i>n</i> = 10−12), sf-LC (green circles, <i>n</i> = 6−7), GlCs (blue circles, <i>n</i> = 4−6), and BC (red circles, <i>n</i> = 6−7). (<i>F</i>) Spike frequency adaptation in response to an injected current (+200 pA, 400 ms-duration). s-GoCs (<i>n</i> = 12), sf-LC (<i>n</i> = 7), and GlCs (<i>n</i> = 6) showed accommodation, but BCs did not (<i>n</i> = 6). The firing frequency adaptation was calculated as the ratio (flast/fin) of the instantaneous frequency at the fourth and last spike intervals in a spike train. *P<0.05, one-way ANOVA with Tukey's post test.</p

Morphological properties of small inhibitory interneurons.

<p>S-GoC: small Golgi cell; sf-LC: small fusiform Lugaro cell; GlC: globular cell. Morphological data were taken from the best preserved small inhibitory interneurons infused with fluorescent dye (s-GoC: <i>n</i> = 14, sf-LC: <i>n</i> = 10, GlC: <i>n</i> = 26). The deformation was estimated by GA/SA. GA: great axis of soma (µm), SA: small axis of soma (µm). Statistical significance was examined using one-way ANOVA with Tukey's post test. > or < indicates statistically significant differences; ≈ indicates no significant difference.</p