Addition of glutamate to serum-free culture promotes recovery of electrical activity in adult hippocampal neurons in vitro

Abstract

A long-term cell culture system utilizing normal adult hippocampal neurons would represent an important tool that could be useful in research on the mature brain, neurological disorders and age-related neurological diseases. Historically, in vitro neuronal systems are derived from embryonic rather than mature brain tissue, a practice predicated upon difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. A few studies have shown that neurons derived from the hippocampal tissue of adult rats can survive and regenerate in vitro under serum-free conditions. However, while the adult neurons regenerated morphologically under these conditions, both the electrical activity characteristic of in vivo neurons as well as long-term neuronal survival was not consistently recovered in vitro. In this study, we report on the development of a defined culture system with the ability to support functional recovery and long-term survival of adult rat hippocampal neurons. In this system, the cell-adhesive substrate, N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, supported neuronal attachment, regeneration, and long-term survival of adult neurons for more than 80 days in vitro. Additionally, the excitatory neurotransmitter glutamate, applied at 25muM for 1-7 days after morphological neuronal regeneration in vitro, enabled full recovery of neuronal electrical activity. This low concentration of glutamate promoted the recovery of neuronal electrical activity but with minimal excitotoxicity. These improvements allowed electrically active adult neurons to survive in vitro for several months, providing a stable test-bed for the long-term study of regeneration in adult-derived neuronal systems, especially for traumatic brain injury (TBI).

Figure 1

Representative phase-contrast and anti-neurofilament/anti-GFAP immunostained pictures of neurons cultured from adult hippocampal tissue. This figure ilustrates both the recovery of the hippocampal neurons in this defined in vitro system as well as the purity of the neuronal culture, neurons versus glial cells. A-I illustrates phase-contrast images of living cultures taken during different culture ages, immediately following cell culture through 28 days in vitro, 40x view. (A) Phase picture of neurons in vitro 1 hour following the attachement of neurons onto a silane-modified coverslip. (B) 6 hours post-attachment (C) 2 days post-attachment. Note the rapid recovery of axons as well as the phase bright cell soma. (D) Phase picture of the neurons after 7 days in vitro. (E) Phase picture of the neurons after 14 days in vitro. Morphologically these adult-derived hippocampal neurons are fully recovered. (F) Phase picture of neurons after 22 days in vitro. (G) Phase picture of neurons after 22 days in vitro, with a prior 1-day exposure to 25 μM glutamate added to the culture media on day 21. (H) Phase picture of neuron after 78 days in vitro. (I) Phase picture of neuron after 78 days in vitro, after incubation with 25 μM glutamate between days 21 to 28, further visualized after application of antibodies against neurofilament (red) and GFAP (green) antibodies (J, K) and (red) MAP-2 (L)

Figure 2

Representative traces for voltage and current clamp of an adult neuron 21 DIV. Neurons retain the ability to move current into and out of cells through the voltage-gated ion channels (voltage clamp trace) as well as to fire single action potentials after electrical stimulation (current clamp trace). These traces originated from adult hippocampal neurons after 21 days in vitro, where 25μM glutamate had been applied to the culture medium between days 20 and 21.

Figure 3

Representative phase-contrast pictures and electrophysiological recordings of adult hippocampal neurons after approximately 80 DIV. (A, B) Phase contrast pictures of neurons 80 DIV. (C) Representative traces for voltage and current clamp of an adult neuron 78 DIV. Neurons retain the ability to move current into and out of cells through the voltage-gated ion channels (voltage clamp trace) as well as to fire single action potentials after electrical stimulation (current clamp trace). These traces originated from adult hippocampal neurons after 78 days in vitro, where 25 μM glutamate had been applied to the culture medium between days 21 and 28.