Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology

Abstract

To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease

“…Primary Neuronal Cultures. Primary neuronal cultures were prepared using a protocol adapted from Rayport et al and previously described (35,47). Briefly, mesencephalic cells containing the substantia nigra pars compacta (excluding the ventral tegmental area) were dissected from coronal sections of brains from postnatal day 1 or 2 rat pups, dissociated in papain, and plated onto glial cells at densities of 4 × 10 5 per coverslip.…”

Amphetamine and Other Weak Bases Act to Promote Reverse Transport of Dopamine in Ventral Midbrain Neurons

“…Methods foi-culture of postnatal ventral midbrain neurons have been described (Rayport et al, 1992). Briefly, rat pups (1-4 days old) were anesthetized with ketamine ( 5 mg intraperitoneally) and the ventral midbrain, encompassing the substantia nigra and the ventral tegmental area, was isolated from a 2-3-mm coronal slice.…”

Neurotrophic Effects of l‐DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures

“…Cultures of ventral midbrain neurons from postnal day 1-2 rats were prepared as in related studies (Rayport et al, 1992;Rayport and Suizer, 1995;Pothos et al, 1996;Przedborski et al, 1996) except that serum-free medium was used as recently described (Mena et al, 1997). Astrocyte monolayers were prepared from the rostral half of the cerebral cortex in ice-cold calcium-and magnesium-free phosphatebuffered saline on postnatal day 2.…”

“…DA neurons in ventral midbrain cultures were identified by TH immunohistochemistry as previously described (Rayport et al, 1992;Przedborski et al, 1996). In brief, cultures were fixed with 4% paraformaldehyde, washed in Tris-buffered saline (0.1 M Tris-HC1 in 9 g/L NaC1, pH 7.4), permeabilized with 0.1% Triton X-100 (Sigma), and incubated at 4°C for 48 h in a 1:640 dilution of monoclonal anti-TH antiserum (Boehringer Mannheim) in Tris-buffered saline containing 0.1% Triton X-100 and 10% normal horse serum.…”