Embryonic Rat Brain Research Articles

DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain

As a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p57Kip2 binds tightly to G1 cyclin/cyclin-dependent kinase complexes to block cell cycle progression. CKIs play critical roles in regulating the transition from proliferation to differentiation in many tissues, including the nervous system. Conversely, CKI dys-regulation contributes to neoplasia and cancer progression. While the combined detection of CKI immunoreactivity and S phase entry using bromodeoxyuridine (BrdU) incorporation may be particularly informative, successful immunostaining may be limited due to "masked" antigen epitopes and acid-induced signal degradation. We now report an improved double immunofluorescent method for detecting p57Kip2 and BrdU in paraformaldehyde-fixed frozen sections of embryonic rat brain. We substituted deoxyribonuclease I (DNAse I) for HCl pre-treatment to expose antigenic sites in frozen sections, and employed a biotinylated tyramide-based system to enhance p57Kip2 visualization. We identified a time- and dose-dependent relationship between DNAse I treatment and double labeling of p57Kip2 and BrdU, increasing both the numbers and intensities of immunopositive nuclei. With excess DNAse I treatment, however, there was signal degradation for both BrdU and total DNA, as reflected by DAPI staining. The use of DNAse I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CKI nuclear factors, and should be useful for both developmental neurobiology studies as well as cancer diagnostic applications.

Arrestin Is Required for Agonist-induced Trafficking of Voltage-dependent Calcium Channels

Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABAB receptor activation, receptors are recruited to the arrestin-channel complex and internalized. beta-Arrestin 1 selectively binds to the SNARE-binding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.

Vasoactive Intestinal Peptide and PACAP38 Control N-Methyl-D-aspartic Acid-induced Dendrite Motility by Modifying the Activities of Rho GTPases and Phosphatidylinositol 3-Kinases

Dendrite morphogenesis is highly dynamic and characterized by the addition and elongation of processes and also by their selective maintenance, retraction, and elimination. Glutamate can influence these events via N-methyl-d-aspartic acid (NMDA) receptors. The neuropeptides vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide-38 (PACAP38) affect neurogenesis and differentiation in the developing nervous system. We report here that the peptides and NMDA acted synergistically on dendrite and branch formation. In stage III hippocampal neurons, NMDA increased not only the addition but also the elimination of new dendrites and branches by activating Rac and Cdc42 and phosphatidylinositol 3-kinases, respectively. When applied alone, the neuropeptides did not influence dendrite or branch formation. However, they reduced the elimination of newly formed dendrites and branches caused by NMDA by preventing the NMDA-induced activation of phosphatidylinositol 3-kinases. This led to the formation of persistent dendrites and branches. Additional timelapse studies on the dynamics of dendrite elongation showed alternating periods of elongation and retraction. Phosphatidylinositol 3-kinases increased the velocities of dendrite elongation and retraction, whereas the neuropeptides prolonged the periods of elongation. By modifying NMDA-induced activation of Rho GTPases and phosphatidylinositol 3-kinases, vasoactive intestinal peptide and PACAP38 could play an important role in the control of dendrite growth and branching during development and in response to neuronal activity.

Survival and differentiation of embryonic neural explants on different biomaterials

Biomaterials prepared from polyacrylamide, ethyl acrylate (EA), and hydroxyethyl acrylate (HEA) in various blend ratios, methyl acrylate and chitosan, were tested in vitro as culture substrates and compared for their ability to be colonized by the cells migrating from embryonic brain explants. Neural explants were isolated from proliferative areas of the medial ganglionic eminence and the cortical ventricular zone of embryonic rat brains and cultured in vitro on the different biomaterials. Chitosan, poly(methyl acrylate), and the 50% wt copolymer of EA and HEA were the most suitable substrates to promote cell attachment and differentiation of the neural cells among those tested. Immunofluorescence microscopy analysis showed that progenitor cells had undergone differentiation and that the resulting glial and neuronal cells expressed their intrinsic morphological characteristics in culture.

Maternal Dietary (n-3) Fatty Acid Deficiency Alters Neurogenesis in the Embryonic Rat Brain

Docosahexaenoic acid [22:6(n-3)] is enriched in brain membrane phospholipids and essential for brain function. Neurogenesis during embryonic and fetal development requires synthesis of large amounts of membrane phospholipid. We determined whether dietary (n-3) fatty acid deficiency during gestation alters neurogenesis in the embryonic rat brain. Female rats were fed diets with 1.3% energy [(n-3) control] or 0.02% energy [(n-3) deficient], from alpha-linolenic acid [18:3(n-3)], beginning 2 wk before gestation. Morphometric analyses were performed on embryonic day 19 to measure the mean thickness of the neuroepithelial proliferative zones corresponding to the cerebral cortex (ventricular and subventricular zones) and dentate gyrus (primary dentate neuroepithelium), and the thickness of the cortical plate and sectional area of the dentate gyrus. Phospholipids and fatty acids were determined by HPLC and GLC. Docosahexaenoic acid was 55-65% lower and (n-6) docosapentaenoic acid [22:5(n-6)] was 150-225% higher in brain phospholipids at embryonic day 19 in the (n-3) deficient (n = 6 litters) than in the control (n = 5 litters) group. The mean thickness of the cortical plate and mean sectional area of the primordial dentate gyrus were 26 and 48% lower, respectively, and the mean thicknesses of the cortical ventricular zone and the primary dentate neuroepithelium were 110 and 70% higher, respectively, in the (n-3) deficient than in the control embryonic day 19 embryos. These studies demonstrate that (n-3) fatty acid deficiency alters neurogenesis in the embryonic rat brain, which could be explained by delay or inhibition of normal development.

Transcription Factor IIAτIs Associated with Undifferentiated Cells and Its Gene Expression Is Repressed in Primary Neurons at the Chromatin Level In Vivo

The levels of General Transcription Factor (TF) IIA were examined during mammalian brain development and in rat embryo fibroblasts and transformed cell lines. The large TFIIA subunit paralogues alphabeta and tau are largely produced in unsynchronized cell lines, yet only TFIIA alphabeta is observed in a number of differentiated tissue extracts. Steady-state protein levels of the TFIIA tau, alphabeta, and gamma subunits were significantly reduced when human embryonal (ec) and hepatic carcinoma cell lines were stimulated to differentiate with either all-trans-retinoic acid (ATRA) or sodium butyrate. ATRA-treated NT2-ec cells required replating to induce a neuronal phenotype and loss of detectable TFIIA tau and gamma proteins. High levels of TFIIA tau, alphabeta, and gamma and Sp factors were identified in extracts from human fetal and rat embryonic day-18 brains, but not in human and rat adult brain extracts. A high histone H3 Lys9/Lys4 methylation ratio was observed in the TFIIA tau promoter of primary hippocampal neurons from day-18 rat embryos, suggesting that repressive epigenetic marks of chromatin prevent TFIIA tau from being transcribed in neurons. We conclude that TFIIA tau is associated with undifferentiated cells during development, yet is down-regulated at the chromatin level upon cellular differentiation.

DCC-dependent Phospholipase C Signaling in Netrin-1-induced Neurite Elongation

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.

Cytosolic labile zinc: a marker for apoptosis in the developing rat brain

Cytosolic zinc accumulation was thought to occur specifically in neuronal death (necrosis) following acute injury. However, a recent study demonstrated that zinc accumulation also occurs in adult rat neurons undergoing apoptosis following target ablation, and in vitro experiments have shown that zinc accumulation may play a causal role in various forms of apoptosis. Here, we examined whether intraneuronal zinc accumulation occurs in central neurons undergoing apoptosis during development. Embryonic and newborn Sprague-Dawley rat brains were double-stained for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) detection of apoptosis and immunohistochemical detection of stage-specific neuronal markers, such as nestin, proliferating cell nuclear antigen (PCNA), TuJ1 and neuronal nuclear specific protein (NeuN). The results revealed that apoptotic cell death occurred in neurons of diverse stages (neural stem cells, and dividing, young and adult neurons) throughout the brain during the embryonic and early postnatal periods. Further staining of brain sections with acid fuchsin or zinc-specific fluorescent dyes showed that all of the apoptotic neurons were acidophilic and contained labile zinc in their cell bodies. Cytosolic zinc accumulation was also observed in cultured cortical neurons undergoing staurosporine- or sodium nitroprusside (SNP)-induced apoptosis. In contrast, zinc chelation with CaEDTA or N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reduced SNP-induced apoptosis but not staurosporine-induced apoptosis, indicating that cytosolic zinc accumulation does not play a causal role in all forms of apoptosis. Finally, the specific cytosolic zinc accumulation may have a practical application as a relatively simple marker for neurons undergoing developmental apoptosis.

120. Tropism and Toxicity of Adeno-Associated Viral Vector Serotypes on Neurons and Glia In Vitro

Viral vector toxicity is a primary concern in choosing an optimal gene delivery vehicle for either studying biological phenomena or evaluating potential therapeutic strategies. Generally, adenoassociated viral vectors are thought to be among the least toxic viral vectors. Currently, over 100 serotypes of AAV have been identified or produced and the tropism of the serotypes varies. In the central nervous system, studies have shown serotypes 1, 2, 5, 6, 7, 8, 9 and 10 are capable of transduction. We have previously reported the transduction of primary neuronal cultures by serotypes 1, 2, 5, and 6. Here, we compare the toxicity of serotypes 1, 2, 5, 6, 7, 8 and 9 and tropism of serotypes 7, 8 and 9 on primary cortical cultures derived from rat embryonic brain tissue. A self-complementing AAV vector containing modified AAV2 terminal repeats flanking a green fluorescent protein expressed from the CMV promoter was packaged using the cap gene from AAV serotypes 1, 2, 5, 6, 7, 8, or 9. All vectors were purified by CsCl centrifugation. Cultures were infected on DIV6 then examined for GFP fluorescence, toxicity and immunoreactivity on DIV8 and 12. At 48 hours after infection at a comparable MOI of 10000, only serotype 1 showed significant toxicity as determined by LDH assay. Using an MTS assay, similar toxicity was observed at an MOI of 100000; however, at a higher MOI, serotypes 1, 6, 7 and 8 exhibited greater toxicity than serotypes 2, 5 and 9. One week after infection, serotypes 7 and 8 resulted in primarily NeuN+ colocalized with GFP fluorescence, however, GFAP+ fibers expressing GFP were detected. Serotype 9 infected both NeuN+ and GFAP+ cells. Overall, AAV serotypes 1, 2, 5, 6, 7, 8 and 9 transduce rat neurons and glia in vitro with varied efficiency and toxicity.

Alterations in cellular phenotypes differentiating from embryonic rat brain neurosphere cultures by immunoselection of neuronal progenitors

The neurosphere culture system is widely used to expand neural stem/progenitor cells in vitro and to provide a source of cells for transplantation approaches to CNS disorders. This study describes the populations of neurones, astrocytes and oligodendrocytes which differentiated from embryonic day (E) 14 rat cortical and striatal tissue grown as neurosphere cultures over three passages. The percentages of cells that adopted neuronal phenotypes decreased with passage, astrocytic percentages increased and oligodendrocytic percentages remained constant. In the second part of this study, immunomagnetic separation was used to positively select neuronal progenitor cells from E14 rat cortical and striatal tissue using an antibody, 2F7, which recognises an epitope on the cell surface of pre- and post-mitotic neurones. These immunomagnetically selected cells were grown as neurosphere cultures over three passages and gave rise to significantly different percentages of neurones, astrocytes and oligodendrocytes than those found in the baseline study. In particular, the percentage of neurones arising from the second and third passages was significantly higher following immunoselection. This indicates that neuronal progenitor cells can be isolated using immunomagnetic separation and then expanded using the neurosphere culture system, to generate enriched populations of neurones that can be used in CNS repair.

Potentiation by high potassium of lipopolysaccharide-induced nitric oxide production from cultured astrocytes

Uptake of K + is an important role of astrocytes to maintain physiological lower extracellular K + concentration in the CNS. In this study, the effect of high K + concentration was examined on the cellular function of astrocytes from embryonic rat brain in primary culture. Nitric oxide (NO) production induced by lipopolysaccharide (LPS) was measured as an index of cellular function of astrocytes. Increasing KCl concentration to about 40 mM did not directly evoke NO production, but doubled the level of LPS (1 ng/ml)-induced NO production. K-gluconate showed a similar enhancing effect although the degree of enhancement was about half of that of KCl. Neither NaCl nor Na-gluconate showed any effect. The K +-channel blocker, 4-aminopyridine, but not tetraethylammonium or apamin, inhibited the enhancing effect of KCl. The LPS-induced iNOS protein expression determined by immunoblotting analysis was enhanced by high K + treatment. The level of iNOS mRNA determined by real-time RT-PCR technique was also augmented by the presence of 40 mM KCl. These results indicate that the elevation of extracellular K + concentration regulates astrocytic cell functions through a mechanism involving K A-type K +-channels and that potentiation of NO production by high K + is due to the augmentation of iNOS mRNA and iNOS protein levels.

LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.

Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons

To determine the impact of aging and 17β-estradiol on neuronal Ca 2+ homeostasis, intracellular Fura-2 Ca 2+-imaging was conducted during 20-pulses of glutamate in hippocampal neurons cultured from embryonic (E18), middle-age (10 months) and old (24 months) rat brain. Marked age-related differences in intracellular Ca 2+ ([Ca 2+]i) homeostasis and striking regulation by 17β-estradiol were seen. Embryonic neurons exhibited the greatest capacity to regulate Ca 2+ homeostasis followed by middle-age neurons. In old neurons, the first peak [Ca 2+]i was substantially greater than at other ages and the return to baseline Ca 2+ rapidly dysregulated with an inability to restore [Ca 2+]i following the first glutamate pulse which persisted throughout the 20 pulses. 17β-Estradiol pretreatment of old neurons profoundly attenuated the peak [Ca 2+]i rise and delayed the age-associated dysregulation of baseline [Ca 2+]i, normalizing responses to those of middle-age neurons treated with estradiol. The efficacy of 17β-estradiol extended below 10 pg/ml with full protection against toxicity from glutamate and Aβ (1–40). These results demonstrate age-associated dysregulation of [Ca 2+]i homeostasis which was largely prevented by 17β-estradiol with implications for estrogen/hormone therapy.

Embryogenesis of the rat telencephalon--a morphologic and stereologic analysis

Comparative researches of borderline between telencephalon neuroepithelium and its surrounding mesenchyme in successive early developing stages lack in literature. The aim of this investigation was to carry out systematic morphologic and stereologic analyses of rat telencephalon in early developmental stages. We analysed semithin (1 microm) serial sections of rat embryonic brain from the 12th (E12) to the 15th (E15) day of gestation. The surface densities (SV) of an external mesenchymal surface, an internal mesenchymal surface and a neuroepithelial (ventricular) surface were examined stereologically and compared. The surface density of the external mesenchymal surface was the biggest at E12 (4,05 mm-1) and the least at E15 (1,87 mm-1)-p<0,0005. The surface density of the internal mesenchymal surface was the biggest at E12 (4,02 mm-1) and the least at E15 (2,69 mm-1)-p<0,0005. The surface density of the internal neuroepithelial surface was the biggest at E12 (3,31 mm-1) and the least at E15 (2,01 mm-1)-p<<0,0005. Our stereological examines give objective numerical proof of significant morphogenetic changes in telencephalon shape described by morphologic analyses. The major advantage of stereological methods is the possibility to carry out the estimation procedures in specified, well-defined brain regions or layers.

Albumin stimulates monocyte chemotactic protein‐1 expression in rat embryonic mixed brain cells

Albumin, a blood protein absent from the adult brain in physiological situations, can be brought into contact with brain cells during development or, in adult, following breakdown of the blood-brain barrier occurring as a result of local inflammation. In the present study, we show that ovalbumin and albumin induce the release of monocyte chemotactic protein 1 (MCP-1/CCL2) from rat embryonic mixed brain cells. A short-term exposure to ovalbumin during the cell dissociation procedure is sufficient to generate MCP-1 mRNA. A comparable effect is observed when the cells are incubated for 4 hr with ovalbumin or rat albumin, while MCP-1 messengers are barely detectable following bovine albumin exposure. The amount of MCP-1 protein measured in 4 hr-supernatants of albumin-treated cells followed the same albumin-inducing pattern as that of MCP-1 mRNA, while all albumins tested induced MCP-1 protein after a 17 hr-incubation period. The albumin-induced MCP-1 production is significantly inhibited in calphostin C-treated cells, suggesting the implication of a protein kinase C-dependent signaling pathway. This MCP-1-inducing activity is maintained after a lipid extraction procedure but abolished by proteinase K or trypsin treatments of albumin. The MCP-1 secretion following albumin contact with nervous cells could thus interfere, by chemotactic gradient formation, with the brain infiltration program of blood-derived cells during development or brain injury.

133. Comparison of Adeno-Associated Viral Vector Serotypes 1, 2, 5 and 6 for Gene Transfer to Neurons and Glia In Vitro

One prominently used viral vector, adeno-associated virus (AAV), successfully transduces cells of the central nervous system in vivo. Studies have compared the in vivo transduction efficiency of several AAV serotypes, primarily 1, 2 and 5. Here, we compare the transduction of cells in primary neuronal cultures derived from rat embryonic brain tissue using the AAV serotypes 1, 2, 5 and 6, purified by CsCI centrifugation. A self-complementing AAV vector containing AAV2 terminal repeats flanking a green fluorescent protein expressed from the CMV promoter was packaged using the cap gene from AAV serotypes 1, 2, 5 or 6. Cultures were treated on DIV6 then examined by immunofluorescence 7 days later. Compared to untreated cultures, all four serotypes did not alter cell viability as measured by cell permeability assays (LDH release and nuclear dye exclusion). At comparable MOIs, serotypes 1 and 6 yielded a higher number of GFP+ cells than types 2 and 5. At an MOI of 20,000, serotypes 1, 2 and 6 expressed GFP in NeuN+ or GFAP+ cells with similar efficiency. At the same MOI, serotype 5 expressed GFP in more GFAP+ than NeuN+ cells. However, with all four serotypes tested, approximately 10|[ndash]|20% of the GFP+ cells were negative for both GFAP and NeuN. These results demonstrate that AAV serotypes 1, 2, 5 and 6 can transduce both neurons and astrocytes in vitro with minimal toxicity and exhibit a similar transduction profile to that observed in vivo.

Novel Alternative Splicing Predicts a Truncated Isoform of the NMDA Receptor Subunit 1 (NR1) in Embryonic Rat Brain

The expression of mesencephalic brain derived neurotrophic factor (BDNF) has been shown to be regulated by dopaminergic neuronal functioning and glutamate receptors (GluRs). In turn, BDNF participates in the regulation of mesencephalic GluRs' expression. In the present study we analyzed, using semi-quantitative RT-PCR, the effect of BDNF as well as of the GluRs agonists NMDA and trans-(+/-)-1-Amino-(1S,3R)-cyclopentane dicarboxylic acid (t-ACPD), on the expression levels of the NMDA GluR subunit 1 (NR1) mRNA, using rat cultured mesencephalic neurons. In the course of this study, a novel rat mRNA splice variant of NR1 was identified. This new NR1 mRNA isoform is characterized by the insertion of an 82 base pair intron containing an inframe stop codon, thus predicting the expression of a putative truncated protein of 465 amino acids. The RT-PCR and in situ hybridization reveals that the novel NR1 mRNA is expressed in various brain regions of the rat embryo, whereas no expression was detected in the adult rat brain. The modulation of the novel NR1 mRNA isoform by both BDNF and the metabotropic GluR agonist t-ACPD, suggests that the resulting putative NR1 truncated protein may be relevant in the regulatory network of glutamatergic neurotransmission in the developing central nervous system.

Soluble CPG15 expressed during early development rescues cortical progenitors from apoptosis

The balance between proliferation and apoptosis is critical for proper development of the nervous system. Yet, little is known about molecules that regulate apoptosis of proliferative neurons. Here we identify a soluble, secreted form of CPG15 expressed in embryonic rat brain regions undergoing rapid proliferation and apoptosis, and show that it protects cultured cortical neurons from apoptosis by preventing activation of caspase 3. Using a lentivirus-delivered small hairpin RNA, we demonstrate that endogenous CPG15 is essential for the survival of undifferentiated cortical progenitors in vitro and in vivo. We further show that CPG15 overexpression in vivo expands the progenitor pool by preventing apoptosis, resulting in an enlarged, indented cortical plate and cellular heterotopias within the ventricular zone, similar to the phenotypes of mutant mice with supernumerary forebrain progenitors. CPG15 expressed during mammalian forebrain morphogenesis may help balance neuronal number by countering apoptosis in specific neuroblasts subpopulations, thus influencing final brain size and shape.

Developmental expression of 5-HT 5A receptor mRNA in the rat brain

In the central nervous system, serotonin (5-HT) may function as a mitogen as well as a neurotransmitter; and its early appearance suggests a potential role in development. The present experiments were done to determine the localization of the mRNA coding for the 5-HT 5A receptor during development of the rat brain. 5-HT 5A gene transcription was assessed by in situ hybridization, from E18 and during postnatal (PN) development. An intense signal of 5-HT 5A mRNA was found in the cerebral cortex and olfactory nucleus at E18, PN0 and PN5. A sharp decrease at PN11 was followed by an increase until reaching the adult level in the cerebral cortex; whereas in the olfactory nucleus, transcription remained weak. In contrast, in the hippocampal formation the signal was weak in the CA1, CA2 and CA3 regions at E18 and PO; increased at P5 and then decreased at P11 before attaining the adult level. We conclude that the gene coding for the 5-HT 5A receptor is already active in the embryonic rat brain and is differentially expressed during development.

Tuning the Elasticity of Biopolymer Gels for Optimal Wound Healing

AbstractSoft polymer networks with large mesh size, not flat rigid surfaces, are the normal environment for most animal cells. Cell structure and function depend on the stiffness of the surfaces on which cells adhere as well as on the type of adhesion complex by which the cell binds its extracellular ligand. Many cell types, including fibroblasts and endothelial cells, switch from a round to spread morphology as stiffness is increased between 1000 and 10,000 Pa. Coincident with the change in morphology are a host of differences in protein phosphorylation levels, expression of integrins, and changes in cytoskeletal protein expression and assembly. In contrast, other cells types such as neutrophils and platelets do not require rigid substrates in order to spread, and neurons extend processes better on soft (50 Pa) materials than on stiffer gels. We compare the stiffness sensing of four cell types: platelets, neurons and astrocytes, a glial cell type derived from embryonic rat brain, and melanoma cells. Astrocytes switch from a round to spread morphology as substrate stiffness increases, but do so over a stiffness range 10 times softer than that over which fibroblasts alter morphology. Stiffness-dependent morphologic changes observed from studies of cells grown on surfaces of protein-laminated polyacrylamide gels that have linear elasticity are also seen when cells are on matrices of natural biopolymers such as fibrin. Biopolymer gels like fibrin can be formed with appropriate stiffness to optimize for neuronal cell survival and patterning, and may have utility for repair of damaged neural tissues. The complex non-linear rheology of fibrin and other gels formed by semi-flexible biopolymers that exhibit strain-stiffening provide additional mechanisms by which cells can respond to and actively remodel the mechanical features of their environment.