Two cell circuits of oriented adult hippocampal neurons on self-assembled monolayers for use in the study of neuronal communication in a defined system

Abstract

In this study, we demonstrate the directed formation of small circuits of electrically active, synaptically connected neurons derived from the hippocampus of adult rats through the use of engineered chemically modified culture surfaces that orient the polarity of the neuronal processes. Although synaptogenesis, synaptic communication, synaptic plasticity, and brain disease pathophysiology can be studied using brain slice or dissociated embryonic neuronal culture systems, the complex elements found in neuronal synapses makes specific studies difficult in these random cultures. The study of synaptic transmission in mature adult neurons and factors affecting synaptic transmission are generally studied in organotypic cultures, in brain slices, or in vivo. However, engineered neuronal networks would allow these studies to be performed instead on simple functional neuronal circuits derived from adult brain tissue. Photolithographic patterned self-assembled monolayers (SAMs) were used to create the two-cell "bidirectional polarity" circuit patterns. This pattern consisted of a cell permissive SAM, N-1[3-(trimethoxysilyl)propyl] diethylenetriamine (DETA), and was composed of two 25 μm somal adhesion sites connected with 5 μm lines acting as surface cues for guided axonal and dendritic regeneration. Surrounding the DETA pattern was a background of a non-cell-permissive poly(ethylene glycol) (PEG) SAM. Adult hippocampal neurons were first cultured on coverslips coated with DETA monolayers and were later passaged onto the PEG-DETA bidirectional polarity patterns in serum-free medium. These neurons followed surface cues, attaching and regenerating only along the DETA substrate to form small engineered neuronal circuits. These circuits were stable for more than 21 days in vitro (DIV), during which synaptic connectivity was evaluated using basic electrophysiological methods.

Figure 1

XPS analysis and metallization reaction for PEG-DETA patterns. (A) XPS survey spectrum of PEG-coated glass coverslip (inset shows high resolution C1s spectrum). (B) XPS survey spectrum of DETA on PEG-coated glass coverslip (inset shows high resolution N1s spectrum) analysis of the two layers. (C) XPS survey spectrum of DETA on ablated PEG-coated glass coverslip. (D) Image of the two-cell circuit bidirectional polarity pattern visualized using palladium catalyzed copper reduction metallization (light lines indicate the DETA regions). Scale bar = 75 μm, line width = 5.5 μm, and somal adhesion site (SAS) = 25 μm.

Figure 2

Time line of the adult hippocampal cell culture process and passage onto PEG-DETA bidirectional polarity patterned coverslips. DIV, days in vitro; DPP, days on polarity patterns.

Figure 3

(A) Time-course pictures of neurons on culture after 4 DIV, 1 DPP, 2 DPP, 6 DPP, 10 DPP, 14 DPP, and 21 DPP. Scale bar = 50 μm. (B) Neuronal conformity to PEG-DETA bidirectional polarity pattern(s). Attachment and regeneration of neurons on the bidirectional polarity patterns was quantified by counting the number of neurons (1) attached to any part of the two-cell circuit pattern and (2) specifically to the SASs. The percentage of neurons attached to the DETA patterns versus the PEG background was quantified (n > 15, where n is the number of patterned coverslips evaluated).

Figure 4

Functional two-cell circuits. NR2A, NR2B, or GluR2/3 (red); synaptophysin (green); neurofilament-M (far-red); and DAPI (blue) expression 14 days after passage in adult neurons on unpatterned DETA-coated control coverslips. Scale bars = 17 μm.

Figure 5

Dual patch clamp recordings were performed on neurons on bidirectional polarity patterns (n = 8, two-cell circuits analyzed) (a). Representative electrophysiological recordings showed both cells were neurons (b): voltage-gated sodium and potassium channels in voltage-clamp experiments. (c) Action potentials generated upon stimulation in current clamp mode where the cells were held at −70 mV. Neuron A, channel 1; neuron B, channel 2. Synaptic connections between the neurons were measured. (d, e) Presynaptic neurons (channel 1) were held at −70 mV, and action potentials were evoked. Postsynaptic currents (channel 2) were measured.