Differential regulation of astrocyte plasminogen activators by insulin-like growth factor-I and epidermal growth factor.

Rat astrocytes synthesize and secrete two types of plasminogen activators (PAs), tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), whose functions are related to cell proliferation, migration, and differentiation during development. The regulation of PAs produced by brain astrocytes is poorly understood. In a previous report we demonstrated that t-PA and u-PA are each independently regulated by cAMP-dependent protein kinase and protein kinase-C. In the present study we examined the effects of three well characterized astrocyte mitogens, insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF), on the PA activities produced and secreted by rat astrocytes in vitro. We found that IGF-I and EGF increase cell-associated total PA activity in astrocyte-conditioned medium (CM). The effects of both growth factors were dose and time dependent, and maximal stimulation was achieved after 72 h of treatment with the highest dose tested (100 nM). IGF-I stimulated the cell-associated PA activity more than the CM activity, whereas EGF showed an opposite pattern, suggesting that the secretion of PA is differentially modulated by IGF-I and EGF. PDGF had no effect on astrocyte PA activities at any dose or time point included in the study. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymography showed type-specific changes in CM and cell-associated PA activity after growth factor treatment. IGF-I stimulated only t-PA, whereas EGF induced a marked increase in u-PA activity and a more limited increase in t-PA. PDGF did not modify either t-PA or u-PA activity. In summary, our results show that IGF-I and EGF each had different effects on PA activities, whereas PDGF had no effect. This diversity in the patterns of growth factor regulation of PAs suggests that the production of astrocyte PAs is not simply related to mitogenesis. More likely, astrocyte PAs are involved in a wide range of growth factor-mediated actions in the developing brain.

Involvement of protein kinase-C in the mitogenic effect of insulin-like growth factor-I on rat astrocytes.

Insulin-like growth factor-I (IGF-I) stimulates the proliferation of many cell types, including astrocytes. Astrocytes are a population of brain cells highly enriched in IGF-I receptors, which unlike neurons, retain the ability to proliferate in the adult brain. Although astrocyte proliferation in response to IGF-I is well documented, the intracellular mechanisms that mediate this phenomenon are poorly defined. Interestingly, activation of protein kinase-C (PKC) by IGF-I has been observed in several cell types. In this report we first characterized the mitogenic effects of IGF-I on highly purified type I rat astrocyte cultures. Next, we determined whether IGF-I activates PKC in our cultures. Finally, since astrocyte proliferation is stimulated by both IGF-I and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA), we decided to test the possible involvement of PKC in the mitogenic activity of IGF-I on astrocytes. IGF-I stimulated the DNA synthesis rate in rat astrocytes. Analysis of the time course revealed that IGF-I (10 nM) induces maximal stimulation of [3H]thymidine incorporation (a 4-fold increase) 16-18 h after exposure. TPA also stimulated mitogenesis in our cultures. The dose-response of [3H]thymidine incorporation induced by IGF-I and TPA indicated that 10 nM was the lowest concentration producing a maximal effect for both agents. Analysis of proteins by Western blot revealed that 10 nM IGF-I translocates PKC(alpha), the predominant PKC isoform in astrocyte cultures, from the cytosol to the membrane fraction within 20 min. A similar activation of PKC was achieved with 100 nM TPA. When astrocytes were exposed to IGF-I (10 nM) and TPA (10 nM) in combination, [3H]thymidine uptake was significantly higher than the uptake induced by either IGF-I (10 nM) or TPA (10 nM) alone. However, the effect of IGF-I plus TPA was not fully additive. In a second experiment, the mitogenic effect of IGF-I was partially abolished in cells depleted of PKC by preincubation with high concentrations of TPA (300 nM). Finally, incubation of astrocytes with the PKC inhibitor H-7 at 20 microM, a concentration that completely blocked the mitogenic action of TPA, only reduced the ability of IGF-I to stimulate DNA synthesis by 50%. In summary, our results demonstrate that IGF-I can rapidly activate PKC in astrocytes, and that PKC activation is involved in the mitogenic effect of IGF-I on these cells. However, we conclude that IGF-I also stimulates astrocyte proliferation through PKC-independent pathways.

Synaptic remodeling in the rat arcuate nucleus during the estrous cycle.

Adult female rats showing regular vaginal cycles were studied in order to determine the number of axosomatic synapses in thin sections of the arcuate nucleus. The number of synapses per length of perikaryal membrane was significantly decreased in estrus, compared to other days of the estrous cycle (P less than 0.05). The reduction in the number of synapses in estrus was accompanied by a decrease in the percentage of the average length of perikaryal membrane covered by presynaptic terminals and by an increase in the percentage of membrane in close apposition of glial processes. Since the average perikaryal perimeter was not significantly changed during the estrous cycle, these results indicate a net decrease in the number of arcuate nucleus axosomatic synapses between proestrus and estrus, with a reinnervation of arcuate neurons between estrus and metestrus. These results suggest that there is a physiological synaptic turnover in the arcuate nucleus of the rat during the estrous cycle.

Estrogen-induced synaptic remodelling in adult rat brain is accompanied by the reorganization of neuronal membranes.

Adult cycling female rats were injected with estradiol valerate (2 mg/100 g body wt.), a treatment which has previously been shown to result in synaptic remodelling in the arcuate nucleus and constant vaginal estrus. During the 32 weeks following estrogen treatment, arcuate nucleus neuronal plasma membranes were quantitatively assessed for intramembrane particle (IMP) number and size using freeze-fracture techniques. Neuronal membranes from untreated cycling females, females injected with oil and untreated males were also studied. Untreated rats had dimorphic sexual phenotypes in membrane organization; female rats had more IMP than males, mainly due to greater numbers of small (less than 10 nm) particles. These sex differences were observed in perikarya and dendritic shafts, but not in dendritic spines. Following estrogen treatment the density of IMP in membranes from females decreased. The IMP changes were found only in neuronal perikarya and dendritic shafts, not in dendritic spines, and were mainly due to a massive decrease in the number of small (less than 10 nm) IMP which was only partially offset by an increase in the number of large (greater than or equal to 10 nm) IMP. Thus, by 32 weeks after estradiol valerate treatment, the number and size of IMP in neuronal membranes from females were not different from those seen in normal males. These results strengthen the idea that estradiol may affect the turnover of certain neuronal membrane components in sex-steroid sensitive areas of the brain.

Estradiol--induced redistribution of glial fibrillary acidic protein immunoreactivity in the rat brain.

The immunohistochemical distribution of the glial fibrillary acidic protein (GFAP), a marker of glial filaments, was studied on coronal sections of the globus pallidus, the area CA4 of the hippocampus and the arcuate nucleus of the hypothalamus, 3 estrogen-sensitive areas of the rat brain. The number and the surface density of the GFAP-immunoreactive cells were evaluated in 6 adult ovariectomized rats injected with a single dose (20 mg/kg) of estradiol valerate (OVX + E2 rats) and in 6 ovariectomized littermates injected with vehicle (OVX rats). Two days after the injection, a similar distribution of the GFAP was observed in the arcuate nucleus of OVX + E2 rats when compared to OVX rats, whereas a significantly (P less than 0.001) increased surface density of GFAP immunoreactive material was observed in the globus pallidus and hippocampus of estradiol-treated rats. Since the number of GFAP-positive cells was unchanged by the estradiol injection, the enhanced surface density of GFAP immunoreactive material in the hippocampus and globus pallidus suggest a possible influence of estradiol on GFAP-immunoreactive glial processes.

Postnatal development of glial fibrillary acidic protein immunoreactivity in the hamster arcuate nucleus.

The postnatal development (from 2 days to 1 year) of glial fibrillary acidic protein (GFAP) immunoreactive cells was studied in the arcuate nucleus of male hamsters. In the first postnatal week, GFAP immunoreactivity was observed in radial glial cells whose cell bodies were located in the ependymal layer. Cell processes of GFAP immunoreactive radial glia crossed the arcuate nucleus and reached the pial surface, where they formed a thin and incomplete external limiting membrane. During the second postnatal week, some immunoreactive cell bodies were also located far from the ependymal layer. Some of these cell bodies presented processes that made contact with the ependymal layer whereas others, probably corresponding to maturing astrocytes, did not show ventricular connections. In the third week, only astrocytes showed GFAP immunoreactive perikarya and their immunoreactive processes reached either the blood vessels to form end-feet, or the basal hypothalamic zone to form the glia limitans. In successive weeks, there was an increase of the amount of GFAP-immunoreactive profiles on the glia limitans and surrounding the arcuate nucleus blood vessels. After the 6th postnatal week we observed some GFAP-immunoreactive cells close to arcuate neurons. The number of these cells increased from the 8th postnatal week. From this age on GFAP immunoreactive astrocytic processes compartimentalized the arcuate nucleus defining several rows of aligned neurons. These results indicate that the cytoarchitectonic organization of GFAP immunoreactive elements and their relationship with neurons, blood vessels and pia is not completed until the first 8 weeks of postnatal life in the arcuate nucleus of the hamster.

The distribution of glial fibrillary acidic protein in the adult rat brain is influenced by the neonatal levels of sex steroids.

Sex steroids during the perinatal period are able to modify the postnatal development of neurons within steroid-sensitive areas in the rat brain. This study was designed to test the possible influence of the early postnatal levels of sex steroids on the morphology of the astrocytes. The experimental manipulation of the neonatal levels of sex steroids was performed by the androgenization of females with a single injection of testosterone propionate and by the orchidectomy of males on the day of birth. Control females received a single injection of vehicle and control males were sham operated. All the animals were sacrificed at 3 months of age postnatally. The immunohistochemical distribution of the glial fibrillary acidic protein (GFAP), a marker of astrocytic filaments, was studied on coronal sections of the dorsal hippocampus, the globus pallidus and the hypothalamic arcuate nucleus. The number of GFAP immunoreactive cells, the number of GFAP immunoreactive primary processes per cell and the surface density of the GFAP immunoreactive material were evaluated. This morphometric evaluation revealed a decreased surface density of GFAP immunoreactive material in the hippocampus, globus pallidus and the ventral part of the arcuate nucleus of orchidectomized males when compared to control males. Sex differences in the distribution of GFAP immunoreactivity were detected in the hippocampus and globus pallidus. These differences were abolished by the androgenization of females. The number of GFAP immunoreactive cells was similar in all the experimental groups, indicating that the differences in surface density represent an effect of sex steroids on the growth of astrocytic processes rather than on the proliferation of astrocytes.

Gap junctions in the hypothalamic arcuate neurons of ovariectomized and estradiol-treated rats.

Freeze-fracture methodology was used to study the organization of the neuronal plasma membrane in the rat arcuate nucleus, an estrogen sensitive area of the hypothalamus. Freeze-fracture replicas were prepared from 6 adult ovariectomized rats injected with a single dose of 17 beta-estradiol and from 6 ovariectomized littermates injected with vehicle. Rats were sacrificed 2 days after the injection. Occasional gap junctions were observed in freeze-fractured neuronal membranes from both groups of animals and their incidence was increased (P less than 0.01) in estradiol treated rats. This study demonstrates gap junctions in arcuate neurons and suggests that these structures may be affected by gonadal hormones.

Sexual differentiation of the neuronal plasma membrane: neonatal levels of sex steroids modulate the number of exo-endocytotic images in the developing rat arcuate neurons.

Exo-endocytotic images and intramembrane protein particles (IMP) were quantitatively assessed in freeze-fracture replicas from the plasma membrane of arcuate neurons of rats aged 0 (newborns), 10, 20 and 100 days postpartum. Membranes contained significantly (P less than 0.02) more IMPs in females than in males. Exo-endocytotic images were increased in newborn and 10-day-old males when compared to adult males or to developing females (48 +/- 6 vs 6 +/- 1 images/100 micron 2 in 10-day-old male and female rats, respectively). Androgenization of females with a single injection of testosterone propionate on the day of birth resulted in an increased number of exo-endocytotic images in developing animals (75 +/- 9 images/100 micron 2, 10-day-old rats) and in the abolishment of the sex differences in the number of IMPs.

Sex differences in plasma membrane concanavalin A binding in the rat arcuate neurons.

Previous studies have shown that synaptic connections and organization of neuronal membranes are sexually dimorphic in the arcuate nucleus of developing and adult rats. These sex differences can be abolished by the perinatal androgenization of females. In this study the label-fracture method of Pinto da Silva and Kan was used in order to determine whether membrane sex differences are related to the glycoconjugates in neuronal plasma membranes. Six Sprague-Dawley female rats treated with testosterone on the day of birth, six control females injected with vehicle and six intact males were studied when they were 100 days old. The arcuate nucleus was dissected and incubated for 2 hours in a solution of 0.25 mg/ml concanavalin A, washed in buffer and incubated for 3 hours in a suspension of horseradish peroxidase-coated colloidal gold. Then, freeze-fracture replicas of the arcuate nucleus were prepared. Colloidal gold labeling was observed to be codistributed with intramembrane particles in the outer membrane face of the neuronal perikaryal plasma membrane. The numerical density of small (less than 10 nm) intramembrane particles and colloidal gold particles was significantly greater in control female membranes when compared to males or to androgenized females. The labeling was significantly reduced when the arcuate nucleus was incubated with concanavalin A in presence of 0.5 M methyl-alpha-manopyranoside. These findings indicate a sex difference in the density and distribution of glycoconjugates and intramembranous particles in the neuronal plasma membrane that is dependent on the perinatal levels of sex steroids and is concordant with, and could be the cause of, sex differences in the pattern of synaptic contacts.

PKCepsilon upregulates voltage-dependent calcium channels in cultured astrocytes.

Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.

Regulation of plasminogen activators and type-1 plasminogen activator inhibitor by cyclic AMP and phorbol ester in rat astrocytes.

Two plasminogen activators (PAs): tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as the type-1 plasminogen activator inhibitor (PAI-1) are synthesized and secreted by rat astrocytes. Preliminary studies suggest that PA activity plays a role in astrocyte development and differentiation. We have examined the regulation of the PA system by the cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in purified rat astrocyte cultures. PKA activity was increased by exposing cultured astrocytes to forskolin or dibutyryl cyclic AMP, whereas PKC activity was stimulated with phorbol-12-myristate 13-acetate (PMA). Activation of both second-messenger pathways produced a time- and dose-dependent increase in the total PA activity. However, based on SDS-PAGE/zymography we found that forskolin increased t-PA activity and reduced u-PA activity, whereas PMA treatment caused a significant increase in u-PA activity without altering t-PA activity. Reverse zymography analysis revealed that astrocyte PAI-1 activity is decreased by forskolin and increased by PMA. Together, these results demonstrate that the components of the PA system in rat astrocytes are independently and reciprocally regulated by PKA and PKC. Our findings raise the possibility that the plasminogen activator system could be involved in some of the actions of growth factors and/or neuromodulators that modulate PKC or PKA in astrocytes.

Possible interaction of the adrenal-gonadal systems on brain catecholamines of adult male rats.

Studies from this laboratory showed that gonadectomy (GDX) alters biogenic amines concentrations in diencephalon during the first 40 days. While the GDX females maintain the differences at day 60, the differences are eliminated in males at that time. In the present work, we have studied in three cerebral regions the adrenal involvement in the mechanism responsible for this normalization of catecholamine concentration in long-term castrated adult male rats. A hypersecretion of adrenal steroids seems to compensate for the lack of gonadal effect when the orchidectomized rats reach adulthood only for diencephalic dopamine.

Influence of sexual differentiation on striatal and limbic catecholamines.

The influence of sexual differentiation of the brain on catecholamine content in the corpus striatum and limbic system was studied. Our results suggest that circulating ovary hormones during the critical period play an important role in the sexual differentiation of dopaminergic neurons in the corpus striatum and limbic system. Absence of androgenic steroids in the critical period leads to permanent alterations in the DA content of the limbic system in the male rat. Gonadectomy does not significantly alter NA levels in either of the two studied brain areas.

rRNA-complementarity in the 5' untranslated region of mRNA specifying the Gtx homeodomain protein: evidence that base- pairing to 18S rRNA affects translational efficiency.

Numerous eukaryotic mRNAs contain sequences complementary to segments of the 18S and 28S rRNAs. Previous studies raised the possibility that these complementarities might allow mRNA-rRNA interactions and affect rates of translation. In the present study, we investigated the mRNA encoding the mouse Gtx homeodomain protein. This mRNA contains within its 5' untranslated region (UTR) a segment that is complementary to two regions of the 18S rRNA, located at nucleotides 701-741 and 1104-1136. A Gtx RNA probe containing this complementarity could be photochemically cross-linked to ribosomal subunits through a linkage to 18S rRNA but not to 28S rRNA. Oligonucleotide-directed RNase H digestion of the rRNA and a reverse transcription analysis localized the cross-linked probe to the complementary segment of 18S rRNA at nucleotides 1104-1136 but not at nucleotides 701-741. To determine whether complementarity in the Gtx mRNA affected translation, a mutational analysis was performed with a Gtx-luciferase fusion construct and four related constructs with altered complementarity to the 18S rRNA. These constructs were examined for their ability to be translated in cell-free lysates prepared from P19 embryonal carcinoma and C6 glioma cell lines and after cellular transfection into these same cell lines. In both cell-free translation and transfection studies, the rate of translation decreased more than 9-fold as the degree of complementarity to nucleotides 1104-1136 of the 18S rRNA increased. We hypothesize that segments complementary to rRNA, such as those contained within the Gtx mRNA, form a category of cis-acting regulatory elements in mRNAs that affect translation by base pairing to rRNA within ribosomes.

Identification and characterization of a RING zinc finger gene (C-RZF) expressed in chicken embryo cells.

To identify changes in gene expression that occur in chicken embryo brain (CEB) cells as a consequence of their binding to the extracellular matrix molecule cytotactin/tenascin (CT/TN), a subtractive hybridization cloning strategy was employed. One of the cDNA clones identified was predicted to encode 381 amino acids and although it did not resemble any known sequences in the nucleic acid or protein data bases, it did contain the sequence motif for the cysteine-rich C3HC4 type of zinc finger, also known as a RING-finger. This sequence was therefore designated the chicken-RING zinc finger (C-RZF). In addition to the RING-finger, the C-RZF sequence also contained motifs for a leucine zipper, a nuclear localization signal, and a stretch of acidic amino acids similar to the activation domains of some transcription factors. Southern analysis suggested that C-RZF is encoded by a single gene. Northern and in situ hybridization analyses of E8 chicken embryo tissues indicated that expression of the C-RZF gene was restricted primarily to brain and heart. Western analysis of the nuclear and cytoplasmic fractions of chicken embryo heart cells and immunofluorescent staining of chicken embryo cardiocytes with anti-C-RZF antibodies demonstrated that the C-RZF protein was present in the nucleus. The data suggest that we have identified another member of the RING-finger family of proteins whose expression in CEB cells may be affected by CT/TN and whose nuclear localization and sequence motifs predict a DNA-binding function in the nucleus.

Rapid effects of gonadal steroids upon hypothalamic neuronal membrane ultrastructure.

Freeze-fracture methodology was used to study rat hypothalamic arcuate nucleus (AN) neuronal plasma membrane organization following in vitro perfusion of brain slices with 17-beta-estradiol (17 beta E2) or other test compounds. Physiological levels (10(-10) M) of 17 beta E2 caused an increase in neuronal membrane exo-endocytotic pits within 1 min of perfusion. The increased density of pits was dose related, sustained at a constant rate during 10 min of perfusion, reverted to control values after perfusion with estradiol-free medium for 1 h, and was accompanied by an increased uptake of horseradish peroxidase by the arcuate nucleus in brain slices. The 17 beta E2-induced increase in exo-endocytotic pit density was blocked by tamoxifen (10(-8) M). Cholesterol (10(-10) M), 17-alpha-estradiol (10(-6) M) or dihydrotestosterone (10(-6) M) had no effect on exo-endocytotic pit density. Testosterone had about 50% the potency of 17 beta E2 in increasing exo-endocytotic pit density. These results indicate that physiological levels of 17 beta E2 can have rapid effects upon arcuate nucleus neuronal membrane ultrastructure.

Nuclear pores in rat hypothalamic arcuate neurons: sex differences and changes during the oestrous cycle.

The numerical density of nuclear pores was assessed on freeze-fracture replicas of hypothalamic arcuate neurons from adult male and female rats. In females the nuclear pore density fluctuated during the oestrous cycle and was higher in oestrus than in pro-oestrus, metoestrus and dioestrus. Nuclear pore density in males and in metoestrus and dioestrus females was similar. The nuclear pore density in male rats was significantly less than that in oestrus and pro-oestrus females. The variation of the number of pores per unit nuclear volume and the total number of pores per nucleus was similar to the variation of the numerical density of nuclear pores. These results provide morphological evidence of cyclic changes in neuronal nucleocytoplasmic traffic during the ovarian cycle.

rRNA complementarity within mRNAs: a possible basis for mRNA-ribosome interactions and translational control.

Our recent demonstration that many eukaryotic mRNAs contain sequences complementary to rRNA led to the hypothesis that these sequences might mediate specific interactions between mRNAs and ribosomes and thereby affect translation. In the present experiments, the ability of complementary sequences to bind to rRNA was investigated by using photochemical cross-linking. RNA probes with perfect complementarity to 18S or 28S rRNA were shown to cross-link specifically to the corresponding rRNA within intact ribosomal subunits. Similar results were obtained by using probes based on natural mRNA sequences with varying degrees of complementarity to the 18S rRNA. RNase H cleavage localized four such probes to complementary regions of the 18S rRNA. The effects of complementarity on translation were assessed by using the mRNA encoding ribosomal protein S15. This mRNA contains a sequence within its coding region that is complementary to the 18S rRNA at 20 of 22 nucleotides. RNA from an S15-luciferase fusion construct was translated in a cell-free lysate and compared with the translation of four related constructs that were mutated to decrease complementarity to the 18S rRNA. These mutations did not alter the amino acid sequence or the codon bias. A correlation between complementarity and translation was observed; constructs with less complementarity increased the amount of translation up to 54%. These findings raised the possibility that direct base-pairing of particular mRNAs to rRNAs within ribosomes may function as a mechanism of translational control.

Neuronal membrane remodelling during the oestrus cycle: a freeze-fracture study in the arcuate nucleus of the rat hypothalamus.

Freeze-fracture methodology was used to study the organization of the neuronal plasma membrane in the rat arcuate nucleus, an oestrogen sensitive area of the hypothalamus. The quantitative evaluation of freeze-fracture replicas of the perikarya, dendritic shafts and dendritic spines revealed that the numerical density of intramembranous particles varied during the ovarian cycle. The number of small (less than 10 nm) particles was decreased, and the number of large (greater than 10 nm) particles was increased, in the P-face of the perikaryal plasma membranes in prooestrus and oestrus when compared to dioestrus. This change was associated with a significant increase in the number of exo-endocytotic images in the perikaryal plasma membrane in prooestrus. Changes in intramembranous particles during the ovarian cycle were not detected in arcuate nucleus dendritic shafts and dendritic spines.