Ascorbate transport by primary cultured neurons and its role in neuronal function and protection against excitotoxicity

Neurons maintain relatively high intracellular concentrations of ascorbic acid, which is achieved primarily by the activity of the sodium-dependent vitamin C transporter SVCT2. In this work, we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate as well as whether this system contributes to maturation of neuronal function and cellular defense against oxidative stress and excitotoxic injury. We found that the SVCT2 helps to maintain high intracellular ascorbate levels, normal ascorbate transport kinetics, and activity-dependent ascorbate recycling. Immunocytochemistry studies revealed that SVCT2 is expressed primarily in the axons of mature hippocampal neurons in culture. In the absence of SVCT2, hippocampal neurons exhibited stunted neurite outgrowth, less glutamate receptor clustering, and reduced spontaneous neuronal activity. Finally, hippocampal cultures from SVCT2-deficient mice showed increased susceptibility to oxidative damage and N-methyl-D-aspartate-induced excitotoxicity. Our results revealed that maintenance of intracellular ascorbate as a result of SVCT2 activity is crucial for neuronal development, functional maturation, and antioxidant responses.     

Loss of glial fibrillary acidic protein results in decreased glutamate transport and inhibition of PKA-induced EAAT2 cell surface trafficking

Loss of the astrocyte-specific intermediate filament protein, glial fibrillary acidic protein (GFAP) results in an increased susceptibility to ischemic insult, enhanced hippocampal LTP, and decreased cerebellar long-term depression (LTD). Because glutamate receptor activation plays a key role in cell death and cellular plasticity responses, we wanted to determine if alterations in glial glutamate transport could contribute to the GFAP null phenotype. To address functional changes in glutamate transport, we measured glutamate uptake in cortical, cerebellar, and hippocampal synaptosomal preparations from age-matched adult wild type and GFAP null mice and demonstrated a 25-30% reduction in the V(max) for d-aspartate uptake in the cortex and hippocampus of GFAP null animals. Western blot analysis of cortical synaptosomal fractions from wild type and GFAP null animals demonstrated that loss of GFAP results in decreases in both astrocytic (EAAT1) and neuronal (EAAT3) glutamate transporter subtypes. Immunohistochemical analysis demonstrated a region-specific modification of neuronal glutamate transporter, EAAT3 trafficking in the GFAP null phenotype. Analysis of primary cortical astrocyte cultures prepared from GFAP null and wild type mice demonstrated that loss of GFAP results in an inability to traffic the glial glutamate transporter, EAAT2, to the surface of the cell following protein kinase A (PKA) stimulation by dibutyryl cAMP. Taken together, these results suggest that the intermediate filament protein, GFAP plays a key role in modulating astrocytic and neuronal glutamate transporter trafficking and function.     

Neuroprotective effects of an adenoviral vector expressing the glucose transporter: a detailed description of the mediating cellular events

Considerable knowledge exists concerning the events mediating neuron death following a necrotic insult; prompted by this, there have now been successful attempts to use gene therapy approaches to protect neurons from such necrotic injury. In many such studies, however, it is not clear what sequence of cellular events connects the overexpression of the transgene with the enhanced survival. We do so, exploring the effects of overexpressing the Glut-1 glucose transporter with an adenoviral vector in hippocampal cultures challenged with the excitotoxin kainic acid (KA). Such overexpression enhanced glucose transport, attenuated the decline in ATP concentrations, decreased the release of excitatory amino acid neurotransmitters, and decreased the total free cytosolic calcium load. Commensurate with these salutary effects, neuronal survival was enhanced with this gene therapy intervention. Thus, the neuroprotective effects of this particular gene therapy occurs within the known framework of the mechanisms of necrotic neuronal injury.     

Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity

The expression of excitatory amino acid transporters (EAATs) in rat hippocampus was studied following kainic acid-induced seizure activity in vivo and in hippocampal slice cultures. Protein and mRNA levels of the glial (EAAT2) and neuronal (EAAT3) transporters were determined with affinity-purified antibodies and oligonucleotide probes, respectively. Kainate treatment decreased EAAT3 immunoreactivity in stratum lacunosum moleculare within 4 h of seizure onset. Upon pyramidal cell death (5 days after kainate treatment), EAAT3 immunoreactivity in stratum pyramidale of CA1 and in stratum lacunosum moleculare was almost completely eliminated. The rapid effect of kainate on EAAT3 expression was confirmed by in situ hybridization; EAAT3 mRNA levels were decreased in CA1 and CA3 regions within 4-8 h of seizure onset. Kainate treatment had an opposite effect on levels and expression of EAAT2. Developmental studies indicated that the rapid regulation of transporter expression was not observed in rats younger than 21 days, an observation congruent with previous reports regarding the resistance of young rats to kainate. In hippocampal organotypic cultures, which lack a major excitatory input from the entorhinal cortex, kainate produced a slow decrease in [3H]d-aspartate uptake. This study indicates that an early effect of kainate treatment consists of down-regulation of the neuronal transporter EAAT3 in restricted hippocampal regions, together with a modest increase in the expression of the glial transporter EAAT2. Differential regulation of neuronal and glial glutamate transporters may thus play a role in kainate-induced seizure, neurotoxicity and neuronal plasticity.     

Differential effects of glucose on dehydroascorbic acid transport and intracellular ascorbate accumulation in astrocytes and skeletal myocytes

Skeletal muscle and brain are major sites of glucose transport and ascorbate (vitamin C) storage. Ascorbate is oxidized to dehydroascorbic acid (DHAA) when used as an enzyme cofactor or free radical scavenger. We evaluated the hypothesis that glucose regulates DHAA uptake and reduction to ascorbate (i.e., recycling) by skeletal muscle cells and cerebral astrocytes. DHAA uptake was inhibited partially by glucose added simultaneously with DHAA. Comparison of wild type L6 skeletal muscle cells with an L6-derived cell line (D23) deficient in facilitative hexose transporter isoform 3 (GLUT3), indicated that both GLUT3 and facilitative hexose transporter isoform 1 (GLUT1) mediate DHAA uptake. Preincubation of muscle cells with glucose inhibited the rates of glucose and DHAA uptake, and decreased the intracellular concentration of ascorbate derived from recycling of DHAA. In contrast, glucose preincubation did not depress GLUT1 protein and activity levels or DHAA recycling in astrocytes. These results establish that glucose downregulates subsequent recycling of DHAA by skeletal muscle cells but not astrocytes.     

Neurotrophic effects of basic and acidic fibroblast growth factors are not mediated through glial cells

Basic and acidic fibroblast growth factors (bFGF, aFGF) increase the survival of fetal hippocampal pyramidal neurons in serum-free cultures. bFGF is also a mitogen for astrocytes either in highly purified glial cultures or as a contaminant in neuronal cultures. The possibility that bFGF enhances neuronal survival indirectly through stimulating glial proliferation is unlikely. In the presence of 1 ng/ml bFGF, the total number of contaminating astrocytes (as defined by immunohistochemical staining for glial fibrillary acidic protein (GFAP] was increased to 4.3% vs 0.9% in control hippocampal cultures. aFGF did not significantly increase astrocyte number while supporting neuronal survival. Two other agents which stimulated equal or greater astrocytic proliferation, epidermal growth factor (EGF) and 10% serum, did not support neurons, and bFGF still significantly increased neuronal survival in their presence. When glial proliferation was inhibited by aphidicolin, contamination decreased to 0.1% in controls and 1.0% with 1 ng/ml bFGF, yet the neurons remained responsive to FGF. Cultures lacking any detectable GFAP-positive cells were identified, and even in the absence of glial cells, aFGF and bFGF increased neuronal survival. Because there is no significant correlation between the neuronal response and astrocyte number, it appears that bFGF and aFGF can directly support neuronal survival.     

Development of beta-adrenergic receptors and their function in glia-neuron communication in cultured chick brain

The beta-receptors of intact neuronal and glial cells of chick embryonic brain were studied via the specific binding of the beta-antagonist [3H]dihydro-L-alprenolol ( [3H]DHA). Cells were cultivated in either highly homogeneous or mixed populations; the neuronal cells were also grown under the influence of glial conditioned medium (GCM) or 10(-11)-10(-10) M L-norepinephrine or L-isoproterenol. The beta-receptors of both neuronal and glial cells proved to be positively cooperative (n = 2.5) and of high affinity, with a Kdapp of 98 and 44 pM, respectively. The Kdapp value was influenced only slightly by the different culture conditions. The receptor concentration was relatively low in the homogeneous neuronal and glial cultures (Bmax = 6.4 and 3.3 fmol/10(6) cells, respectively). It increased by a factor of 2-3 if development of the neuron-glia contacts in the culture was possible (mixed cultures). GCM and beta-agonists elevated the number of beta-receptors of the neuronal cells approximately 4-fold, even in the absence of glial cells. This receptor-number change was preceded by a well observable morphological differentiation. Both the morphological and the beta-receptor effects of L-norepinephrine were antagonized by L-propranolol. The beta-receptor number increased about 2-fold during a 10-day in vitro development, even in neuron-glia mixed cultures.     

Dexamethasone and forskolin synergistically increase [Met]enkephalin accumulation in mixed brain cell cultures

Possible synergistic effects of the glucocorticoid dexamethasone (DEX, 10⁻⁷ M) and the adenylate cyclase agonist forskolin (FSK, 10⁻⁵ M) on [Met⁵]enkephalin (ME) accumulation were examined in enriched rat glial cultures and in mixed neuronal/glial cultures. In enriched glial cultures, DEX and FSK each stimulated the accumulation of ME 2–3-fold over basal media levels, but there was little additional stimulation when these agonists were combined. In contrast, mixed neuronal/glial cultures showed only weak responses to DEX or FSK alone, but the combination of these agonists produced a pronounced synergistic effect on media ME accumulation (6–10-fold over basal levels). The DEX effect was mediated via a classical glucocorticoid receptor, since DEX was potent (acting over a concentration range of 10⁻¹¹−10⁻⁷ M), mimicked by corticosterone (10⁻⁶ M), and blocked by the glucocorticoid receptor antagonist RU486. There was a pronounced time lag (2 days) for the synergistic effects of DEX+FSK to develop. In situ hybridization and immunocytochemical studies suggested that astrocytes were the major source for the increased ME production in all mixed neuronal/glial cultures examined. Creating a mixed culture by plating fetal neurons onto confluent, enriched P7 glial cultures inhibited accumulation of ME in the media. DEX+FSK, but neither agonist alone, overcame this neuronal inhibition and increased accumulation of media ME to levels identical to levels in stimulated enriched glial cultures. The net effect was a 6-fold increase in ME accumulation in the mixed neuronal/glial cultures relative to a 2.5-fold increase in the enriched glial cultures. Neuronal inhibition of basal glial ME production could explain the similar synergistic effects of DEX+FSK observed in all mixed neuronal/glial cultures examined, and may be important in suppressing ME production by astrocytes in the brain.     

GABA uptake by purified avian Müller glia cells in culture

GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na(+)/Cl (-) dependent transporters (GATs). It has been reported in the past that, in the avian retina, [(3)H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [(3)H]GABA in a Na(+) and Cl(+) dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [(3)H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K[(+) or veratridine stimulation, Müller glial cells did not release [(3)H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.     

A new neuromodulatory pathway with a glial contribution mediated via A(2a) adenosine receptors

A low concentration (10 nM) of adenosine potentiated hippocampal neuronal activity via A(2a) adenosine receptors without affecting presynaptic glutamate release or postsynaptic glutamatergic conductance. Adenosine inhibited glutamate uptake through the glial glutamate transporter, GLT-1, via A(2a) adenosine receptors. In addition, adenosine stimulated GLT-1-independent glutamate release from astrocytes, possibly in response to a rise in intracellular Ca(2+), via A(2a) adenosine receptors involving PKA activation. Those adenosine actions could lead to an increase in synaptic glutamate concentrations responsible for the potentiation of hippocampal neuronal activity. The results of the present study thus represent a novel neuromodulatory pathway with a glial contribution, bearing both inhibition of GLT-1 function and stimulation of glial glutamate release, as mediated via A(2a) adenosine receptors.     

Neuronal dependency of the glycine transporter GLYT1 expression in glial cells

Two membrane-localized transporter proteins (GLYT1 and GLYT2) are responsible for removal of extracellular glycine in the mammalian CNS. Whereas GLYT1 seems to be expressed mainly in glial cells, GLYT2 is neuronal. The highest concentrations of both transporters are found in glycinergic areas of the nervous system. The expression of these proteins may be under regulatory control. We demonstrate here that GLYT1 is not expressed in pure glial cultures, but it is expressed by diverse types of glial cells in mixed neuronal/glial cultures. In these mixed cultures, the glial expression of GLYT1 is down-regulated after selective elimination of the neurons. The absence of expression in pure glial cultures and the observed reduction in the GLYT1 expression after neuronal loss support the existence of a regulatory cross-talk between neurons and glia to initiate and sustain the glial expression of GLYT1. GLIA 20:155–162, 1997. © 1997 Wiley-Liss Inc.     

Culturing conditions determine neuronal and glial excitability

The cultivation of pure neuronal cultures is considered advantageous for the investigation of cell-type specific responses (such as transmitter release and also pharmacological agents), however, divergent results are a likely consequence of media modifications and culture composition. Using Fura-2 based imaging techniques, we here set out to compare calcium responses of rat hippocampal neurones and glia to excitatory stimulation with l-glutamate in different culture types and media. Neurones in neurone-enriched cultures had increased responses to 10 μM and 100 μM l-glutamate (+43 and 45%, respectively; p's< 0.001) and a slower recovery compared to mixed cultures, indicating heightened excitability. In matured (15-20 days in vitro) mixed cultures, neuronal responder rates were suppressed in a neurone-supportive medium (Neurobasal-A, NB: 65%) compared to a general-purpose medium (supplemented minimal essential medium, MEM: 96%). Glial response size in contrast did not differ greatly in isolated or mixed cultures maintained in MEM, but responder rates were suppressed in both culture types in NB (e.g. 10 μM l-glutamate responders in mixed cultures: 29% in NB, 71% in MEM). This indicates that medium composition is more important for glial excitability than the presence of neurones, whereas the presence of glia has an important impact on neuronal excitability. Therefore, careful consideration of culturing conditions is crucial for interpretation and comparison of experimental results. Especially for investigations of toxicity and neuroprotection mixed cultures may be more physiologically relevant over isolated cultures as they comprise aspects of mutual influences between glia and neurones.     

Glutamine and glutamate transport in cultured neuronal and glial cells

The uptake of L-glutamine in neuronal and glial cultures derived from rat cerebral hemispheres was found to be mediated by a low affinity-high capacity mechanism which was concentrative in both cell types; the calculated Km and Vmax were twice as high in glial than in neuronal cultures. In contrast L-glutamate was taken up by a high affinity system which was particularly efficient and concentrative in the glial cells. Different transport mechanisms for L-glutamine appeared to operate in the two cell types: L-glutamine uptake in neurons was sodium-dependent, specifically inhibited by L-glutamine but not affected by high potassium concentrations in the external medium; on the other hand, glial glutamine transport was decreased when potassium concentration increased, was sodium-independent and significantly inhibited by 3 structurally related amino acids. No significant contribution of homoexchange could be detected in either cell type. After [14C]glutamine preincubation, the radioactivity released into the superfusion medium by neuronal cells was increased in the presence of a high potassium concentration; no such effect could be seen in the case of glial cultures. A regulatory mechanism is suggested where astrocyte depolarization and repolarization would channel a flux of glutamine toward the neurons, subsequent to a glutamate flux in the opposite direction.     

Localization of brain endothelial luminal and abluminal transporters with immunogold electron microscopy

Immunogold electron microscopy has identified a variety of blood-brain barrier (BBB) proteins with transporter and regulatory functions. For example, isoforms of the glucose transporter, protein kinase C (PKC), and caveolin-1 are BBB specific. Isoform 1 of the facilitative glucose transporter family (GLUT1) is expressed solely in endothelial (and pericyte) domains, and ∼75% of the protein is membrane-localized in humans. Evidence is presented for a water cotransport function of BBB GLUT1. A shift in transporter polarity characterized by increased luminal membrane GLUT1 is seen when BBB glucose transport is upregulated; but a greater abluminal membrane density is seen in the human BBB when GLUT1 is downregulated. PKC colocalizes with GLUT1 within these endothelial domains during up- and downregulation, suggesting that a PKC-mediated mechanism regulates human BBB glucose transporter expression. Occludin and claudin-5 (like other tight-junctional proteins) exhibit a restricted distribution, and are expressed solely within interendothelial clefts of the BBB. GFAP (glial fibrillary acidic protein) is uniformly expressed throughout the foot-processes and the entire astrocyte. But the microvascular-facing membranes of the glial processes that contact the basal laminae are also polarized, and their transporters may also redistribute within the astrocyte. Monocarboxylic acid transporter and water channel (Aquaporin-4) expression are enriched at the glial foot-process, and both undergo physiological modulation. We suggest that as transcytosis and efflux mechanisms generate interest as potential neurotherapeutic targets, electron microscopic confirmation of their site-specific expression patterns will continue to support the CNS drug discovery process.     

Different effects of monocarboxylates on neuronal survival and beta-amyloid toxicity

Glucose is a principal metabolic fuel in the central nervous system, but, when glucose is unavailable, the brain can utilize alternative metabolic substrates such as monocarboxylates to sustain brain functions. This study examined whether the replacement of glucose with monocarboxylates (particularly pyruvate and lactate) had an equivalent effect of glucose on neuronal survival in rat hippocampal organotypic slice cultures, or ameliorate the neurotoxicity induced by amyloid beta-peptide (Abeta). The possible mechanism was also explored. We found that pyruvate and lactate alone increased cell death in the hippocampal slice cultures at 24 and 48 h. Supplementation of glucose-containing culture media and Abeta-treated culture media with pyruvate, but not lactate, attenuated cell death as strong as with trolox, known as a reactive oxygen species scavenger, and niacinamide, an NAD(+) precursor, and this protective effect was reversed by alpha-cyano-4-hydroxycinnamic acid. Pyruvate significantly increased the aconitase activity and the NAD(+) levels in the hippocampal slices in the presence of Abeta, but did not maintain the ATP levels. Our results indicate that pyruvate and lactate alone cannot replace glucose as an alternative energy source to preserve the neuronal viability in the hippocampal slice cultures. Pyruvate, in the presence of glucose, improves neuronal survival in the hippocampal slice cultures and also protects neurons against Abeta-induced cell death in which mitochondrial NAD(P) redox status may play a central role.     

Antipsychotic drugs influence transport of the beta-adrenergic antagonist [3H]-dihydroalprenolol into neuronal and blood cells.

The amine hypothesis suggests that the cause of schizophrenic or depressive psychosis is dysfunction of noradrenergic or serotonergic neurotransmission. We investigated pharmacological properties of [3H]-dihydroalprenolol (DHA) transport into C6, IMR32, native lymphocytes, B-lymphoblastoids and MOLT-3 cells. DHA transport was inhibited by a heterogeneous group of structurally related compounds exhibiting an amine group and various aromatic ring structures. It was verified on cells of neuronal/glial and blood cell origin but in detail on B-lymphoblastoids. The latter once showed strongest inhibition of DHA transport using tricyclic antidepressants (amitriptyline: IC50 = 2.86 microM, imipramine: IC50 = 3.33 microM) and haloperidol (IC50 = 3.98 microM) as a neuroleptic. Antipsychotics like clozapine (IC50 = 11 microM), olanzapine (IC50 = 15 microM), spiperone (IC50 = 66 microM) and EMD 49980 (ICso > 100 microM) were less effective. In contrast to cells of blood origin, a stimulation of DHA transport by antipsychotics was not detectable using neuronal cells. As antipsychotics showed a distinct inhibition and, concerning cells of blood origin, a stimulation of transport after pre-incubation, further investigations seem to be of interest in respect to its involvement in the cellular uptake of drugs and therefore its impact on the quality of therapy of psychiatric patients.     

Magnesium deprivation decreases cellular reduced glutathione and causes oxidative neuronal death in murine cortical cultures

The vulnerability of cultured cortical neurons to oxidative injury is an inverse function of the extracellular Mg2+ concentration. In order to test the hypothesis that depolarization-enhanced release of reduced glutathione (GSH) contributes to this phenomenon, we assessed the effect of Mg2+ deprivation on cellular and medium glutathione levels. Incubation of mixed neuronal and glial cultures in Mg2+-free medium resulted in a decline in cellular total glutathione (GSx) within 8 h, without change in oxidized glutathione (GSSG); no effect was seen in pure glial cultures. This decrease in cellular GSx was associated with a progressive increase in GSx but not GSSG in the culture medium. Cellular GSH loss was not attenuated by concomitant treatment with antioxidants (ascorbate, Trolox, or deferoxamine), but was prevented by the NMDA receptor antagonist MK-801. Mg2+ deprivation for over 24 h produced neuronal but not glial death, with release of about 40% of neuronal lactate dehydrogenase by 48-60 h. Most of this cytotoxicity was prevented by treatment with either antioxidants or MK-801. These results suggest that Mg2+ deprivation causes release of neuronal reduced glutathione via a mechanism involving excessive NMDA receptor activation. If prolonged, cellular GSH depletion ensues, leading to oxidative neuronal death.     

Dearth of glutamate transporters contributes to striatal excitotoxicity

Since excitotoxicity is hypothesized to contribute to cell death in Huntington's disease (HD), we examined the susceptibility of striatal and hippocampal neurons to glutamate-induced cell death. Striatal cultures were more susceptible to glutamate-triggered toxicity than sister hippocampal cultures. Dose-response curves were equivalent when secondary toxicity was blocked with application of the NMDA receptor antagonist, MK801, or enhanced with the pan-specific glutamate transport blocker, TBOA, following excitotoxin removal. TBOA failed to alter the dose-response characteristics of striatal excitotoxicity, ruling out reverse operation of glutamate transporters. Striatal cultures expressed less EAAC1 and less membrane-associated EAAC1, GLT1, and GLAST than hippocampal cultures. Antisense down-regulation of EAAC1 increased the sensitivity of hippocampal cultures to glutamate, indicating that this transporter can act as an important neuroprotectant. Thus, the relative expression levels of glutamate transporters, even in parts of the brain where they are considered adequately expressed, appear to influence the sensitivities of different neuronal populations to excitotoxicity.     

A rapid method to quantify neurons in mixed cultures based on the specific binding of [3H]ouabain to neuronal Na+,K(+)-ATPase.

The high-affinity binding of [3H]ouabain to Na+,K(+)-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [3H]ouabain was found to be time-dependent, high-affinity (apparent Kd = 8.5 nM), saturable (Bmax = 20.6 pmol/mg protein), dependent upon the presence of ATP, inhibited by K+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins N-methyl-D-aspartate (NMDA) and kainate, reduced the amount of [3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [3H]ouabain binding to the neuronal form of Na+,K(+)-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.     

A primary culture system for biochemical analyses of neuronal proteins

Low-density cultures of embryonic rat hippocampal neurons have been widely used to investigate localization and function of neuronal proteins using immunocytochemistry and electrophysiology. These cultures provide a relatively homogeneous population of hippocampal pyramidal neurons and interneurons compared to post-natal mixed neuron/glial cultures from hippocampus, cerebral cortex, and cerebellum. However, the limited quantity of neurons and the difficulty in harvesting adequate amounts makes biochemical analyses of endogenous neuronal proteins in these low-density cultured neurons difficult. Here, we provide detailed methods to prepare cultures of embryonic rat hippocampal neurons suitable for biochemical analyses of both endogenously and exogenously expressed proteins. The procedures described here are also suitable for comprehensive studies of expression, localization, post-translational modification, and function of neuronal proteins in the same neuronal culture system.