Temporal neurotransmitter conditioning restores the functional activity of adult spinal cord neurons in long-term culture

Abstract

The ability to culture functional adult mammalian spinal cord neurons represents an important step in the understanding and treatment of a spectrum of neurological disorders including spinal cord injury. Previously, the limited functional recovery of these cells, as characterized by a diminished ability to initiate action potentials and to exhibit repetitive firing patterns, has arisen as a major impediment to their physiological relevance. In this report, we demonstrate that single temporal doses of the neurotransmitters serotonin, glutamate (N-acetyl-DL-glutamic acid) and acetylcholine-chloride lead to the full electrophysiological functional recovery of adult mammalian spinal cord neurons, when they are cultured under defined serum-free conditions. Approximately 60% of the neurons treated regained their electrophysiological signature, often firing single, double and, most importantly, multiple action potentials.

Figure 1

Outline of the defined culture system to study the regeneration of adult mammalian spinal cord neurons.

Figure 2

Immunocytochemical evidence of the early events during the initiation of the regeneration process utilizing nestin and neurofilament-150. Upper panel. The regeneration process was initiated during the first 24 hours of cell plating and the live/dead assay indicates the majority of the plated cells are alive. Lower panel. Early regeneration events are characterized by co-expression of the nestin and neurofilament 150 proteins by most neurons between day 1–3. By day 4, the neurons only express neurofilament-150 and other neuron specific markers, as the nestin expression was lost by day 4.

Figure 3

Immunostained cultures at day 35 utilizing different neuron specific antibodies. A. Phase coupled with fluroscence micrograph showing neurons stained with ISLET-1 antibody (a putative motoneuron marker). B. Fluroscent staining of the ISLET-1 positive cells shown in figure A. C. Neurons stained with MO-1 antibody (a putative motoneuron marker). D. Neurons stained with ChAT antibody (a putative motoneuron marker). E. Neurons double-stained with MAP 2a and b and NF 150 antibodies. F. Neurons double-stained with synaptophysin and NF 150 antibodies. G. Neurons stained with NF 150 antibody. H. Neurons stained with MAP 2, a and b antibody.

Figure 4

Representative phase-contrast pictures of the cells which were used to quantify the electrical properties. A and B. Phase pictures of the neurons in control culture at day 44. C. Phase pictures of the neurons after glutamate treatment at day 37 (G37). D. Phase pictures of the neurons after glutamate treatment at day 44 (G44). E. Phase pictures of the neurons after serotonin treatment at day 37 (S37). F. Phase pictures of the neurons after serotonin treatment at day 44 (S44). G. Phase pictures of the neurons after acetylcholine chloride treatment at day 37 (A37). H. Phase pictures of the neurons after acetylcholine chloride treatment at day 44 (A44). I. Phase pictures of the neurons after glutamate+serotonin treatment at day 37 (GS37). J and K. Phase pictures of the neurons after glutamate+serotonin treatment at day 44 (GS44). L, M, N and O. Phase pictures of the neurons after glutamate+serotonin followed by acetylcholine chloride treatment at day 44 (GSA44).

Figure 5

Electrophysiological recordings from glutamate+serotonin→ acetylcholine chloride (GSA44) treated cultures. A. Scheme for single neurotransmitters application. B. Scheme for multiple neurotransmitters application. C. Representative trace for voltage and current clamp of a neuron firing a single action potential after multiple neurotransmitter applications at day 44. D. Representative trace for voltage and current clamp recordings of a double action potential firing neuron after multiple neurotransmitter applications at day 44. E. Representative trace for voltage and current clamp recordings of a neuron firing multiple action potentials after multiple neurotransmitter applications at day 44.

Figure 5

Electrophysiological recordings from glutamate+serotonin→ acetylcholine chloride (GSA44) treated cultures. A. Scheme for single neurotransmitters application. B. Scheme for multiple neurotransmitters application. C. Representative trace for voltage and current clamp of a neuron firing a single action potential after multiple neurotransmitter applications at day 44. D. Representative trace for voltage and current clamp recordings of a double action potential firing neuron after multiple neurotransmitter applications at day 44. E. Representative trace for voltage and current clamp recordings of a neuron firing multiple action potentials after multiple neurotransmitter applications at day 44.

Figure 5

Electrophysiological recordings from glutamate+serotonin→ acetylcholine chloride (GSA44) treated cultures. A. Scheme for single neurotransmitters application. B. Scheme for multiple neurotransmitters application. C. Representative trace for voltage and current clamp of a neuron firing a single action potential after multiple neurotransmitter applications at day 44. D. Representative trace for voltage and current clamp recordings of a double action potential firing neuron after multiple neurotransmitter applications at day 44. E. Representative trace for voltage and current clamp recordings of a neuron firing multiple action potentials after multiple neurotransmitter applications at day 44.

Figure 5

Electrophysiological recordings from glutamate+serotonin→ acetylcholine chloride (GSA44) treated cultures. A. Scheme for single neurotransmitters application. B. Scheme for multiple neurotransmitters application. C. Representative trace for voltage and current clamp of a neuron firing a single action potential after multiple neurotransmitter applications at day 44. D. Representative trace for voltage and current clamp recordings of a double action potential firing neuron after multiple neurotransmitter applications at day 44. E. Representative trace for voltage and current clamp recordings of a neuron firing multiple action potentials after multiple neurotransmitter applications at day 44.

Figure 5

Electrophysiological recordings from glutamate+serotonin→ acetylcholine chloride (GSA44) treated cultures. A. Scheme for single neurotransmitters application. B. Scheme for multiple neurotransmitters application. C. Representative trace for voltage and current clamp of a neuron firing a single action potential after multiple neurotransmitter applications at day 44. D. Representative trace for voltage and current clamp recordings of a double action potential firing neuron after multiple neurotransmitter applications at day 44. E. Representative trace for voltage and current clamp recordings of a neuron firing multiple action potentials after multiple neurotransmitter applications at day 44.