The identity of Hb9-eGFP expressing neurons in mouse spinal cord sections
In spinal cord sections of E12-14 embryos and early postnatal Hb9-eGFP mice, numerous cells throughout ventral spinal cord express eGFP (, E13, P1). Only large Hb9-eGFP cells in ventral spinal cord express motoneuron marker ChAT, but not small cells (, E13, P1). Approximately 90% of ChAT-positive neurons in developing ventral horn are Hb9-eGFP-positive.
Figure 1 Hb9-eGFP-positive neurons in spinal cord sections of embryonic, early postnatal, and adult Hb9-eGFP transgenic mice. A, In embryonic day 13 (E13), postnatal day 1 (P1), and adult Hb9-eGFP transgenic mice, low-power fluorescent micrographs illustrate the (more ...)
In adult mouse spinal cord sections, eGFP expression is mostly restricted to ventral spinal cord in lamina VIII and IX (, Adult). A few Hb9-eGFP+ cells were also observed near the central canal in the intermediomedial cell column and lamina X (, Adult). The majority (~96%) of Hb9-eGFP+ cells in ventral horn are large, ChAT-positive cells (, Adult) that are surrounded by GlyT2- and GAD-positive boutons (). About half (~55%) of the large ChAT-positive neurons in adult mouse spinal cord ventral horn are Hb9-eGFP-positive (, Adult). In medial lamina VIII, intermediomedial cell column, and lamina X, subsets of small Hb9-eGFP+ neurons were also observed (, Adult). They were neither calbindin-positive nor ChAT-positive ().
Identification of motoneurons in Hb9-eGFP mouse spinal cord cultures
In dissociated spinal cord cultures obtained from E12-14 embryos, Hb9-eGFP+ cells are divided into three groups according to their somal sizes at DIV 12-16: large- (diameter > 28 μm), medium- (diameter 10-28 μm), and small- (diameter < 10 μm) sized cells. Cells in cultures were double-stained with motoneuron markers SMI-32 and ChAT. Of the large-sized Hb9-eGFP cells, 98.2 ± 2.1 % (n = 68) were double-labeled with SMI-32- and ChAT-immunoreactivities. These SMI-32/ChAT- positive large Hb9-eGFP cells had characteristic motoneuron morphology with a fusiform, triangular, or polygonal soma, a prominent dendritic arborization, at least one broad dendrite whose diameter rivaled that of the soma and emanates from the pole of the cell, frequently branching into thinner secondary processes, and generally a single long axon-like neurite, often extending over several millimeters (). The percentage of medium-sized Hb9-eGFP cells that were SMI-32/ChAT-positive was 42.7 ± 3.2% (n = 224), and these cells had characteristic morphologies that mimic the large-sized Hb9-eGFP cells (). Of the small-sized Hb9-eGFP cells, 96.9 ± 1.2 % (n = 98) were SMI-32-/ChAT-, with an oval or round soma and bipolar or multiple thin dendrites emerging from the soma (, arrow). 3.1 ± 1.2 % (n = 98) small-sized Hb9-eGFP cells were SMI-32+ and/or ChAT+, which are likely to be ChAT+ interneurons or Hb9-eGFP+ interneurons (data not shown). Therefore, numerous living motoneurons in the dissociated spinal cord cultures were identified reliably by their Hb9-eGFP expression, size, and characteristic morphology. The surfaces of eGFP+ putative motoneurons were decorated with neurotransmitter markers GlyT2- and GAD-positive boutons in the dissociated spinal cord cultures (), demonstrating glycinergic and GABAergic presynaptic innervations.
Figure 2 Identification of motoneurons in Hb9-eGFP mouse spinal cord cultures. A, Hb9-eGFP+ neurons in DIV 12-16 dissociated spinal cord cultures co-labeled with SMI-32 (red) and ChAT (blue). Large eGFP+ cells (Ai-Aii) that are double-labeled with SMI-32- and (more ...)
Glycine-induced currents are decreased in G93A-SOD1 motoneurons
Using whole-cell patch clamp recording technique, we examined the electrophysiological properties of GlyRs in cultured Hb9-eGFP motoneurons (). Experiments were performed in the presence of the voltage-dependent sodium channel blocker TTX (0.5 μM), the GABAA
R antagonist bicuculline (5 μM), and non-NMDA and NMDA ionotropic glutamate receptor antagonists CNQX (5 μM) and APV (50 μM), respectively. Bath application of 5 μM bicuculline was reported to block the majority of GABAA
R-mediated responses but with little effect on GlyR-mediated responses (O'Brien and Berger, 1999
). Pressure ejection was used to apply 100 ms pulses (1-2 psi) of 1 mM glycine onto Hb9-eGFP+
neurons. In 61 of 64 Hb9-eGFP motoneurons, the glycine pulses produced large inward currents when the membrane potential was held at -65 mV (mean current density = 118.1 ± 8.2 pA/pF; ). Glycine-induced small inward currents (< 20 pA/pF) were seen in 3 of 64 cells. In small-sized Hb9-eGFP neurons, the peak amplitudes of glycine-evoked currents were highly variable, with the current densities range from ~ 50 pA/pF to ~ 150 pA/pF (n
= 15). Only Hb9-eGFP motoneurons with large inward currents were analyzed in the following experiments comparing responses in control and G93A-SOD1 motoneurons. The inward current was completely blocked by bath application of GlyR antagonist strychnine (1 μM; ), indicating that the glycine-induced responses are mediated by GlyR activation.
Figure 3 Glycine-induced currents are decreased in G93A-SOD1 motoneurons. A, An Hb9-eGFP+ motoneuron is identified under fluorescent microscope, and recorded under differential interference contrast (DIC) scope, along with a patch electrode (right) and a drug (more ...)
To examine GlyRs in ALS mouse motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and recorded glycine-evoked currents in cultured Hb9-eGFP motoneurons prepared from G93A-SOD1 embryos and from their non-mutant SOD1 transgenic littermates. In each group, Hb9-eGFP motoneurons with characteristic morphologies were randomly selected and recorded. At equivalent days in culture, G93A-SOD1 motoneurons were slightly larger in size (membrane capacitance = 43.5 ± 2.4, n = 20) than control motoneurons (membrane capacitance = 39.8 ± 1.8, n = 17), but no significant difference was detected. Pressure ejection of glycine at -65 mV produced an inward current markedly smaller in the G93A-SOD1 motoneurons than in the control motoneurons (). The decrease of glycine-evoked current density was significant as evidenced by the ~35% reduction in G93A-SOD1 motoneurons compared with control motoneurons (control: 121.2 ± 9.5 pA/pF, n = 20; G93A-SOD1: 78.6 ± 4.9 pA/pF, n = 17). No significant difference was observed in the rise and decay time of the glycine-evoked currents between control and G93A-SOD1 motoneurons (data not shown).
Current-voltage relationships for glycine-evoked currents were also examined in control and G93A-SOD1 motoneurons (). Measurements of the glycine-evoked currents at holding potentials ranging from -70 to +50 mV produced an approximately linear current-voltage relationship. G93A-SOD1 motoneurons had decreased peak amplitude of glycine currents at all holding potentials, but no significant difference was observed in the reversal potential of glycine-evoked currents ().
Glycinergic miniature postsynaptic currents are altered in G93A-SOD1 motoneurons
The data so far demonstrate that glycine-induced currents are decreased in ALS mouse motoneurons. However, GlyRs could differ in functional properties and numbers at the synapse compared with non-synaptic receptors. To directly examine synaptic GlyRs in spinal cord cultures, we performed an analysis of glycinergic mIPSCs. mIPSCs represent postsynaptic responses to the spontaneous release of single quanta of neurotransmitter and can therefore be used to detect changes in synaptic receptor properties without the complicating presynaptic variables of action potential propagation and evoked calcium influx into the presynaptic terminal. We recorded glycinergic mIPSCs in Hb9-eGFP motoneurons voltage-clamped at -65 mV in the presence of TTX (0.5 μM), CNQX (5 μM), APV (50 μM), and bicuculline (5 μM). Under this condition, fast rising and fast decaying spontaneous synaptic currents were detected in most Hb9-eGFP motoneurons (55 of 62) (), reflecting in our motoneuron culture model the local release of glycine from presynaptic terminals as determined subsequently by immunofluorescence studies using antibodies against GlyT2 and synaptophysin (). The peak amplitude of these mIPSCs ranged from -5 to -620 pA, and occurred at a frequency of 0.21 ± 0.03 Hz. Perfusion of the GlyR antagonist strychnine (1 μM) abolished the responses (), confirming that these events are mediated by GlyRs.
Figure 4 Glycinergic miniature postsynaptic currents (mIPSCs) are altered in G93A-SOD1 motoneurons. A, Glycinergic mIPSCs are detected in Hb9-eGFP motoneurons in the presence of tetrodotoxin (TTX; 0.5 μM), 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX; 5 (more ...)
Figure 8 Surface glycine receptor (GlyR) expression is decreased in G93A-SOD1 motoneurons. A, Confocal images show the localization of GlyRs (Ai-Aiii, red) in close apposition to postsynaptic scaffold protein gephyrin (Geph; Ai, blue)-, presynaptic GlyT2- (Aii, (more ...)
The average amplitude of glycinergic mIPSCs were significantly smaller in the G93A-SOD1 motoneurons than in the control (), with the mean current densities of mIPSCs for control and G93A-SOD1 motoneurons were 2.48 ± 0.25 pA/pF (n
= 20) and 1.79 ± 0.19 pA/pF (n
= 17), respectively. Cumulative amplitude histograms for mIPSCs showed a shift towards smaller amplitudes for G93A-SOD1 motoneurons (). The percentage of small-amplitude mIPSCs (< 20 pA) in G93A-SOD1 motoneurons (22.7 ± 2.3 %, n
= 17) was significantly higher than in the control motoneurons (14.3 ± 1.6 %, n
= 20). The decay phase of mIPSC events was slightly faster in G93A-SOD1 than in the control motoneurons (), with decay times of 21.33 ± 2.18 ms for G93A-SOD1 and 25.51 ± 2.59 ms for control, respectively, but no significant difference was detected. The frequency of mIPSC events in G93A-SOD1 motoneurons (0.13 ± 0.02 Hz, n
= 17) was lower than in the control motoneurons (0.21 ± 0.03 Hz, n
= 20), but no significant difference was detected (p
= 0.09; Supplemental Figure 1
). No significant differences in the rise time of mIPSC events were observed between control and G93A-SOD1 motoneurons (data not shown).
GlyR currents are rescued in G93A-SOD1 motoneurons from chimeric cultures
We also designed motoneuron recording experiments using spinal cord chimeric cultures composed of mixtures of normal (wild-type) cells and cells that express G93A-SOD1, eg. a culture prepared from mixtures of Hb9-eGFP/G93A-SOD1 embryonic spinal cords and their Hb9-eGFP/non-mutant SOD1 transgenic littermate spinal cords. Individual motoneurons were harvested after recordings were made and were genotyped by single-cell PCR for the human SOD1 gene after whole genome amplification (, top). Surprisingly, no significant alternations were observed in glycine-induced current densities between G93A-SOD1/Hb9-eGFP motoneurons (127.2 ± 7.0 pA/pF, n = 8) and control motoneurons (130.7 ± 11.9 pA/pF, n = 7; ). The amplitude of glycinergic mIPSCs was not different between G93A-SOD1 motoneurons and control motoneurons (), with the mean current densities of mIPSCs for G93A-SOD1 and control motoneurons were 2.24 ± 0.21 pA/pF (n = 8) and 2.39 ± 0.26 pA/pF (n = 7), respectively. This indicates a neural network involvement in the ultimate decline of motoneuron GlyR physiology and that wild-type neurons can compensate for network failure.
Figure 5 Glycine receptor-mediated currents are rescued in G93A-SOD1 motoneurons in chimeric culture. A, top, Single-cell genotyping of a non-mutant SOD1/Hb9-eGFP motoneuron and a G93A-SOD1/Hb9-eGFP motoneuron from the same culture. Bottom, glycine-evoked currents (more ...)
GABAA receptor currents are not affected in G93A-SOD1 motoneurons
GABAAR-mediated currents were also examined in Hb9-eGFP motoneurons. In the presence of TTX (0.5 μM), CNQX (5 μM), APV (50 μM), and strychnine (0.5 μM), brief pulses of GABA (100 μM, 100 ms, 1-2 psi) evoked inward currents in 30 of 37 Hb9-eGFP motoneurons (mean current density = 49.8 ± 6.8 pA/pF). The current was reversibly abolished by bath application of bicuculline (50 μM), a competitive antagonist of GABAARs, thus indicating that the current was mediated by GABAAR activation (). The peak amplitude of GABA-induced currents were slightly higher in G93A-SOD motoneurons than in control motoneurons, but no significant differences were observed (mean current densities for control and G93A-SOD were 49.8 ± 6.8 pA/pF, n = 30 and 51.4 ± 4.6 pA/pF, n = 24, respectively; ). No significant differences were observed in the decay time of GABA-induced currents in G93A-SOD1 and control motoneurons (decay time for control and G93A-SOD1 were 3.94 ± 0.29 s, n = 30, and 4.63 ± 0.43 s, n = 24, respectively).
Figure 6 GABAA receptor-mediated currents are not affected in G93A-SOD1 motoneurons. A, A 100 ms pulse (2-3 psi) of GABA (100 μM) evokes an inhibitory current that is blocked by bicuculline (Bic; 50 μM). B, Sample recordings of GABA-evoked currents (more ...)
GABAergic mIPSCs were detected in 29 of 37 Hb9-eGFP motoneurons in the presence of an antagonist mixture containing TTX (0.5 μM), CNQX (5 μM), APV (50 μM), and strychnine (0.5 μM). The mean current densities of GABAergic mIPSCs was smaller (0.228 ± 0.031 pA/pF, n = 29), and the decay time phase was slower (59.02 ± 7.21 ms, n = 29) than that of glycinergic mIPSCs. These mIPSCs were blocked by bicuculline (50 μM), suggesting that they were mediated by activation of GABAARs (). No significant differences existed in the peak amplitude of the mIPSCs between control and G93A-SOD1 motoneurons (mean current densities for control and G93A-SOD1 were 0.228 ± 0.031 pA/pF, n = 29, and 0.231 ± 0.041 pA/pF, n = 24, respectively; ). No significant differences in the frequency, rise and decay times of GABAergic mIPSC events were observed between control and G93A-SOD1 motoneurons (data not shown).
GlyR mRNA expression is decreased in G93A-SOD1 motoneurons
To determine the molecular mechanisms underlying the GlyR current reduction in G93A-SOD1 motoneurons, we used quantitative single-cell real-time RT-PCR to examine GlyR mRNA expression in G93A-SOD1 and control Hb9 motoneurons. GlyRs isolated from mammalian spinal cord are pentameric membrane proteins composed of α and β subunits (Pfeiffer et al., 1982
; Prior et al., 1992
). GlyRs exhibit subtype heterogeneity due to four isoforms of α subunit (α1- α4) (Kuhse et al., 1995
; Lynch, 2009
). GlyRs in immature neurons contain the abundantly expressed α2 subunit, but GlyRs in mature neurons contain predominantly the α1 subunit within 2 weeks after birth (Malosio et al., 1991
). The α3 subunit is mainly expressed in the cerebellum (Malosio et al., 1991
) and the α4 subunit is a murine gene that is not expressed in human (Matzenbach et al., 1994
). Because the majority of glycinergic neurotransmission in adults is mediated by α1β GlyRs and because we evaluated motoneurons that were morphologically well-differentiated, only the α1 subunit (Glra1) was analyzed in this study. A housekeeping gene β-actin
was used as an endogenous control (Calvo et al., 2008
). The standard curves for the GlyRα1 and β-actin show approximately the same slope (), indicating similar amplification efficiency (Halford, 1999
; Medhurst et al., 2000
). GlyRα1 mRNA was detected in 45 of 47 (96%) individual Hb9-eGFP motoneurons. shows amplification plots of GlyRα1 transcripts obtained from two individual Hb9-eGFP motoneurons (one from a control and one from a G93A-SOD1 culture). Ct
values were obtained for GlyRα1 and β-actin (indicated by the bold line). Ct
values for β-actin were similar in the two motoneurons (). In contrast, GlyRα1 transcript amount in the G93A-SOD1 motoneuron was lower than that of the control motoneuron, because a higher Ct
number was necessary to reach the threshold (). The data were normalized to β-actin levels and statistic analysis of the ΔCt
revealed a significant difference between control motoneurons (ΔCt
= 3.06 ± 0.16; n
= 15) and G93A-SOD1 motoneurons (ΔCt
= 4.27 ± 0.15; n
= 28). The calibration of ΔCt
values gives the relative amount of GlyRα1 transcripts in the respective motoneuron groups (). G93A-SOD1 motoneurons had lower GlyRα1 mRNA levels compared to control motoneurons (), suggesting that downregulation of GlyRα1 gene expression may underlie insufficient functioning of G93A-SOD1 motoneurons. To determine if the reduction in GlyRα1 mRNA was motoneuron specific, a subset of small bipolar Hb9 interneurons (, inset
) were also examined. No significant change was observed in GlyRα1 mRNA expression in small bipolar Hb9 interneurons between control and G93A-SOD1 cultures (), indicating that the decrease in GlyRα1 mRNA level is specific to motoneurons.
Figure 7 Glycine receptor (GlyR) α1 subunit mRNA expression is decreased in G93A-SOD1 motoneurons. A, A validation experiment showing threshold cycles (Ct) for GlyRα1 (squares) and β-actin (triangles) plotted against total spinal cord mRNA (more ...)
Surface GlyRs are decreased in G93A-SOD1 motoneurons
To validate the mRNA expression data obtained from single-cell RT-PCR, we used immunocytochemistry to detect GlyRα1 subunit protein in cultured Hb9-eGFP motoneurons. The localization of GlyR was examined by confocal microscopy after labeling receptors with an anti-GlyR antibody and an antibody against gephyrin, the anchoring protein of GlyR (Triller et al., 1985
; Betz et al., 1994
). GlyR and gephyrin cluster postsynaptically at glycine-releasing nerve terminals (Triller et al., 1985
; Triller et al., 1987
). At DIV14, GlyR and gephyrin staining was localized in clusters at the surface of the soma and dendrites of Hb9-eGFP motoneurons (), and they were almost perfectly colocalized (colocalization coefficients M1 = 0.946 ± 0.012, n
= 6; calculated as M1 = pixelsCh1, coloc
where Ch1 is GlyR). Most GlyR clusters on the surface of soma and dendrites of Hb9-eGFP motoneurons were associated with presynaptic GlyT2-boutons (colocalization coefficients M1 = 0.917 ± 0.019, n
= 6; calculated as M1 = pixelsCh1, coloc
where Ch1 is GlyR), suggesting a close association with glycinergic presynaptic terminals (). Surface GlyR cluster labeling and gephyrin staining were also observed in close apposition to presynaptic synaptophysin (colocalization coefficients M1 = 0.911 ± 0.015, n
= 6; calculated as M1 = pixelsCh1, coloc
where Ch1 is GlyR; M1 = 0.922 ± 0.014, n
= 6; calculated as M1 = pixelsCh1, coloc
where Ch1 is gephyrin), a ubiquitous synaptic vesicle marker (Navone et al., 1986
), demonstrating directly the existence of glycinergic synaptic connections in this cell culture model (). Intracellular labeling of GlyRs was also detected within the soma, mostly around the nucleus (). The intracellular GlyR-immunoreactivity did not colocalize with gephyrin, GlyT2, or synaptophysin (), suggesting that these are extrasynaptic receptors. No attempts were made to study intracellular GlyRs. Surface GlyRs were detected in 47 of 56 Hb9-eGFP motoneurons, whereas the staining of GlyRs in small-sized Hb9-eGFP neurons varies: some have very few GlyR staining, while some have very intense GlyR staining (data not shown). These are consistent with the electrophysiological results.
We examined the number of clusters on the surface of soma and proximal dendrites, which mainly represent the functional postsynaptic GlyRs. GlyR cluster number on the soma and proximal dendrites of G93A-SOD1/Hb9-eGFP motoneurons was significantly lower than control motoneurons () (control: 29.9 ± 1.6 for soma and 37.4 ± 1.1 for proximal dendrites, n = 47; G93A-SOD1: 24.5 ± 1.1 for soma and 31.7 ± 1.6 for proximal dendrites, n = 42, respectively). Receptor alternation patterns were similar in the soma and proximal dendrites ().