Interneuronal synapses formed by motor neurons appear to be glutamatergic

Abstract

Acetylcholine release at motor neuron synapses has been long established; however, recent discoveries indicate that synaptic transmission by motor neurons is more complex than previously thought. Using whole-cell patch clamp, we show that spontaneous excitatory postsynaptic currents of rat motor neurons in primary ventral horn cultures are entirely glutamatergic, although the cells respond to exogenous acetylcholine. Motor neurons in cultures express the vesicular glutamate transporter VGlut2, and culturing motor neurons for weeks with glutamate receptors blocked upregulates glutamate signaling without increasing cholinergic signaling. In spinal cord slices, motor neurons showed no decrease in spontaneous excitatory synaptic potentials after blocking acetylcholine receptors. Our results suggest that motor neuron synapses formed on other neurons are largely glutamatergic in culture and the spinal cord.

Figure 1. Immunolabeling of primary motor neuron cultures

Cultured spinal motor neurons are stained with antibodies against the motor neuron marker Hb9, the vesicular glutamate transporter VGlut2. Secondary antibodies (Jackson ImmunoResearch Laboratories) were labeled with DyLight 549 (red) or FITC (green) from. DAPI staining (blue) indicates cell nuclei. “Merge” combines the left two panels in each row. Images taken after 6 days in culture.

Figure 2. Synaptic transmission in ventral horn cultures is entirely glutamatergic

Top: Sample traces showing sEPSCs recorded from motor neurons in 14-day-old cultures with ACh antagonists (10 μM each atropine and mecamylamine; left) and a glutamatergic antagonist (10 μM CNQX; right). Bottom: Pooled data showing average sEPSC frequency and amplitude from 5 motor neurons in each condition. (p < 0.01, One-way ANOVA).

Figure 3. Cultured motor neurons show excitatory responses to ACh

A. Sample traces of action potentials (APs) recorded in current clamp mode from cultured motor neurons. Notice the depolarization and increase in EPSP frequency stimulated by ACh in the trace 2^nd from bottom. B. Top: Average AP frequency for motor neurons that increased their firing in response to ACh (n = 6; * p < 0.05; one way ANOVA). Bottom. Average AP frequency for motor neurons that did not increase their firing rate (n = 5). Notice the overall low action potential frequency. C. ACh (100 μM) was added alone or in the presence of 10 μM atropine to block muscarinic receptors or 10 μM mecamylamine to block nicotinic receptors.

Figure 4. Chronic block of glutamate receptors upregulates glutamate signaling, but does not increase cholinergic signaling

Motor neuron cultures were grown in glutamate/glutamine-free media and treated 20 μM CNQX plus 100 μM AP5 for 14 days after establishing culture. “Control media” - cells grown in standard neurobasal media without glutamate antagonists. “Glutamatergic blocking media” – cells grown with glutamate antagonists. No antagonists were present during the recording. “CNQX +AP5 during recording” – cells grown with glutamate antagonists in which the same antagonists were added back during the recording. A. Sample traces of sEPSCs. B. Sample traces of miniature EPSCs. C. Average frequency of EPSCs for control medium (n = 8) and blocking media (n = 9; * = p < 0.05 compared to control media; One way ANOVA).

Figure 5. Synaptic activity of motor neurons in spinal cord slices not affected by ACh receptor block

Top: Sample traces of sEPSCs recorded from motor neurons in spinal cord slices from 6 -7 day-old rat pups with and without ACh receptor antagonists atropine + mecamylamine (10 μM each). Bottom: Pooled data averaged for 8 cells. A + M added for 5 minutes before recording (p > 0.05, One way ANOVA).