Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

<p>Abstract</p> <p>Background</p> <p>Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.</p> <p>Results</p> <p>To develop a new tool for the identification of <it>in vitro </it>enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (<it>penk1</it>) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the <it>penk1 </it>promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors.</p> <p>Conclusions</p> <p>These results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The <it>penk1</it>-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.</p

Syntaxin 1B, but not syntaxin 1A, is necessary for the regulation of synaptic vesicle exocytosis and of the readily releasable pool at central synapses.

Two syntaxin 1 (STX1) isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t)-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission

Properties of evoked EPSCs in STX1B^−/− neurons.

<p>(A) Representative mean eEPSC waveforms in WT and STX1B^−/− synapses (left). Neurons were stimulated at 25 ms intervals. Amplitude of AMPA receptor-mediated eEPSCs of STX1B^−/− (<i>n</i> = 18) and WT (<i>n</i> = 22) neurons (right). Scale bar: 50 ms. (B) Paired-pulse ratio of EPSCs was significantly different in cultured neurons from STX1B^−/− and WT mice (WT, <i>n</i> = 19; STX1B^−/−, <i>n</i> = 14; <i>p</i> = 0.049, one-way repeated measure ANOVA with two sample <i>t</i>-test). (C) Amplitude of individual eEPSCs during the stimulus train triggered by 200 stimuli at 1 Hz from STX1B^−/− (<i>n</i> = 7) and WT (<i>n</i> = 16) mice, normalized to the amplitude of first response and plotted as a function of stimulus number. (D) Representative eEPSC train stimulated by 20 Hz, recorded from a representative WT (top) and STX1B^−/− (bottom) neuron. The first (left) and last (right) twenty responses are presented. Stimulus artifacts were truncated. Scale bar: 0.5 s, 0.5 nA. (E) Amplitude of individual eEPSCs during the stimulus train triggered by 200 stimuli at 20 Hz. A significant difference between neurons from STX1B^−/− and WT mice was observed (WT, <i>n</i> = 7; STX1B^−/−, <i>n</i> = 8; <i>p</i><0.001, one-way repeated measure ANOVA). (F) Mean cumulative EPSC amplitude value from 40 stimuli, 20 Hz trains in WT and STX1B^−/− neurons. Data points between stimuli 30 and 40 (top) and 180 and 200 (bottom) were fitted by linear regression and back-extrapolated to time 0 to estimate the vesicle pool size.</p

Deletion of STX1B decreases the size of RRP in glutamatergic and GABAergic synapses.

<p>(A) Average EPSC evoked by application of 0.5 M hypertonic sucrose solution for 15 s in WT and STX1B^−/− neurons (left). Mean RRP size estimated from integral charge transfer induced by application of 0.5 M sucrose solution (right; WT, 2.27±0.17 nC, <i>n</i> = 88; STX1B^−/−, 1.39±0.14 nC, <i>n</i> = 62; <i>p</i><0.001, two sample <i>t</i>-test). Scale bar: 10 s, 100 pA. (B) Plot of the integral charge transfer as a function of the frequency of mEPSCs. Lines indicate linear fits through data points for each genotype. Although their slopes were not significantly different, the intercept was significantly lower in STX1B^−/− neurons (<i>p</i> = 0.006, ANCOVA). (C) Average IPSC evoked by application of 0.5 M sucrose solution in WT and STX1B^−/− neurons (left). Mean RRP size estimated from integral charge transfer induced by application of 0.5 M sucrose solution (right; WT, 4.72±0.45 nC, <i>n</i> = 48; STX1B^−/−, 1.45±0.28 nC, <i>n</i> = 35; <i>p</i><0.001, two sample <i>t</i>-test). Scale bar: 10 s, 0.5 nA. (D) Plot of the integral charge transfer as a function of the frequency of mIPSCs. Lines indicate linear fits through data points for each genotype. Although their slopes were not significantly different, the intercept was significantly decreased in STX1B^−/− neurons (<i>p</i><0.001, ANCOVA). *** <i>p</i><0.001.</p

Deletion of STX1B decreases frequency of mEPSCs and mIPSCs.

<p>(A) Representative mean mEPSC waveforms in WT and STX1B^−/− synapses. Scale bar: 10 ms. (B) Representative mEPSCs. Scale bar: 1 s, 100 pA. (C) Cumulative fraction of the distribution of mEPSC frequency. Frequency of mEPSCs was significantly lower in STX1B^−/− neurons (<i>n</i> = 33; WT: <i>n</i> = 45; <i>p</i><0.001, Kolmogorov-Smirnov test). (D) Cumulative fraction of the distribution of mEPSC amplitude. Mean amplitudes of mEPSCs are 36.9±0.30 pA and 37.9±0.55 pA in WT and STX1B^−/−, respectively. (E) Representative mean mIPSC waveforms in WT and STX1B^−/− synapses. Scale bar: 40 ms. (F) Representative mIPSCs. Scale bar: 1 s, 100 pA. (G) Cumulative fraction of the mIPSC frequency distribution. Frequency of mIPSCs was significantly lower in STX1B^−/− neurons (<i>n</i> = 52; WT: <i>n</i> = 29; <i>p</i><0.001, Kolmogorov-Smirnov test). (H) Cumulative fraction of the mIPSC amplitude distribution. Average amplitudes of mIPSCs: WT, 53.38±0.48 pA; STX1B^−/−, 50.49±0.44 pA.</p

Properties of evoked IPSCs in STX1B null neurons.

<p>(A) Representative mean eIPSC waveforms in WT and STX1B^−/− synapses (left). Neurons were stimulated at 25 ms interval. Calcium dependence of eIPSCs (right). Average amplitudes of eIPSCs from STX1B^−/− (<i>n</i> = 6) and WT (<i>n</i> = 9) mice were monitored in various concentrations of free extracellular calcium. Scale bar: 50 ms. (B) Paired-pulse ratio of IPSCs was significantly different in cultured neurons from STX1B^−/− and WT mice (WT, <i>n</i> = 22; STX1B^−/−, <i>n</i> = 20; <i>p</i> = 0.008, one-way repeated measure ANOVA with two sample <i>t</i>-test). (C) The amplitude of individual eIPSCs during the stimulus train triggered by 200 stimuli at 1 Hz from STX1B^−/− (<i>n</i> = 6) and WT (<i>n</i> = 8) mice, normalized to the amplitude of first response and plotted as a function of stimulus number. (D) Representative eIPSCs train stimulated by 20 Hz recorded from a WT (top) and STX1B^−/− (bottom) neuron. The first (left) and last (right) twenty responses are presented. Stimulus artifacts were truncated. Scale bar: 0.5 s, 200 pA. (E) The amplitudes of individual eIPSCs during the stimulus train triggered by 200 stimuli at 20 Hz were significantly different between neurons from STX1B^−/− and WT mice (WT, <i>n</i> = 6; STX1B^−/−, <i>n</i> = 5; <i>p</i> = 0.045, one-way repeated measure ANOVA). (F) Mean cumulative IPSC amplitude value from 40 stimuli, 20 Hz trains in WT and STX1B^−/− neurons. Data points between stimuli 30 and 40 (top) and 180 and 200 (bottom) were fitted by linear regression and back-extrapolated to time 0 to estimate the vesicle pool size.</p

STX1B is required for maintenance of Munc18-1 and vesicle docking.

<p>(A) Western blot analysis of STX1A, STX1B and other presynaptic proteins in STX1B^+/− and STX1B^−/− mice. Whole-brain homogenate was prepared from P7 mice. (B) Expression levels of Munc18-1, STX1A and STX1B from STX1 mutant mice (WT, <i>n</i> = 3; STX1B^+/−, <i>n</i> = 5; STX1B^−/−, <i>n</i> = 4; STX1A^−/−, <i>n</i> = 4; one-way ANOVA with Tukey's test). (C) Electron micrographs of the 7-day-old hippocampus of WT and STX1B^−/− synapses. Scale bar: 100 nm. (D) Number of synaptic vesicles per µm² of presynaptic terminal in WT and STX1B^−/− synapses (WT, <i>n</i> = 55; STX1B^−/−, <i>n</i> = 54; <i>p</i> = 0.19, two sample <i>t</i>-test). (E) The number of docked synaptic vesicles per µm of active zone length. The number of docked vesicles was significantly lower in STX1B^−/− synapses than those of WTs (WT, <i>n</i> = 15; STX1B^−/−, <i>n</i> = 14; <i>p</i> = 0.006, two sample <i>t</i>-test). (F) Distribution of SVs. The distribution was significantly different at 0–50 nm from active zone in STX1B^−/− synapses (WT, <i>n</i> = 15; STX1B^−/−, <i>n</i> = 14; <i>p</i> = 0.03, two sample <i>t</i>-test). (G) Synapse density in hippocampal CA1 region (WT, <i>n</i> = 55; STX1B^−/−, <i>n</i> = 54; <i>p</i><0.001, two sample <i>t</i>-test).* <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001.</p

Properties of neurotransmitter release in DKO neurons.

<p>(A) Representative mean mEPSC waveforms in WT and DKO synapses. Scale bar: 10 ms. (B) Representative mean mIPSC waveforms in WT and DKO synapses. Scale bar: 40 ms. (Ci-ii) Representative autaptic eEPSC in DKO neurons. Triangles indicate asynchronous component of transmitter release. Stimulus artifacts were truncated. Scale bar: 20 ms, 400 pA. (Di-ii) Representative autaptic eIPSC in DKO neurons. Triangles indicate asynchronous component of transmitter release. Stimulus artifacts were truncated. Scale bar: 20 ms, 200 pA.</p