Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.     

Lamina-specific synaptic connections of hippocampal neurons in vitro

By using slice cultures as a model, we demonstrate here that different target selectivities exist among the various afferent fibers to the hippocampus. As in intact animals, septohippocampal cholinergic fibers, provided by a slice culture of septum, innervate a co-cultured slice of hippocampus diffusely, that is, without forming distinct layers of termination. As in vivo, the septal cholinergic fibers establish synapses with a variety of target cells. Conversely, fibers from an entorhinal slice co-cultured to a hippocampal slice display their normal laminar specificity. They preferentially terminate in the outer molecular layer of the fascia dentata, thereby selectively contacting peripheral dendrites of the granule cells. This preferential termination on peripheral dendritic segments is remarkable, since these fibers do not have to compete with commissural fibers, hypothalamic fibers, and septal afferents for dendritic space under these culture conditions. Moreover, in triplet cultures in which first two hippocampal slices were co-cultured and then, with a delay of 5 days, an entorhinal slice was added, the fibers from the entorhinal slice and those from the hippocampal culture terminated in their appropriate layers in the hippocampal target culture. However, in this approach the normal sequence of ingrowth of these two afferents was reversed. In normal ontogenetic development, entorhinal afferents arrive in the hippocampus before the commissural fibers. The results show that there are different degrees of target selectivity of hippocampal afferents and that the characteristic lamination of certain afferent fibers in the hippocampus is not determined by their sequential ingrowth during development. © 1995 John Wiley & Sons, Inc.     

Parvalbumin Immunoreactivity and Protein Level are Altered in the Gerbil Hippocampus During Normal Aging

Hippocampal interneurons are local circuit neurons which are responsible for inhibitory activity in the hippocampus. Parvalbumin (PV) is one of useful markers for GABAergic interneurons, not for principle cells, in the hippocampus. In the present study, we investigated age-related changes in PV immunoreactive neurons and protein levels in the gerbil hippocampus during normal aging. PV immunoreactive neurons were detected in all hippocampal subregions of all groups. PV immunoreactive neurons, which innervated principal neurons, were non-pyramidal neurons in the hippocampal CA1-3 regions, and were polymorphic neurons in the dentate gyrus. In the hippocampal CA1 region, the number of PV immunoreactive neurons was significantly reduced in the postnatal month 3 (PM 3) group, which was sustained by PM 18, and, at PM 24, the number of PV immunoreactive neurons was significantly decreased. In the CA2/3 region and dentate gyrus, the number of PV immunoreactive neurons was significantly decreased at PM 6: Thereafter, the number of PV immunoreactive neurons was sustained until PM 24. In addition, changes in PV protein levels in the gerbil hippocampus were similar to immunohistochemical changes during normal aging: PV protein levels were significantly decreased with age by PM 6: Thereafter, PV protein levels were sustained by PM 24. These results suggest that PV immunoreactive interneurons were decreased in the hippocampus with age in gerbils.     

Demonstration of muscarinic acetylcholine receptor-like immunoreactivity in the rat forebrain and upper brainstem

Summary The distribution of muscarinic acetylcholine receptor protein (mAChR) in the rat forebrain and upper brainstem was described by using a monoclonal antibody (M35) raised against mAChR purified from bovine forebrain homogenates. A method is investigated for light microscopic (LM) and electronmicroscopic (EM) immunocytochemical visualization of reactivity to mAChR-proteins. Putative cholinoceptive neurons including their dendrites were found immunoreactive in the cortical mantle, hippocampus, basal ganglia, amygdala, thalamus and several midbrain regions. In the neocortex, immunoprecipitate with M35 was mainly present in layer 5 pyramidal cells, some layer 3 pyramidal neurons and layer 2 stellate cells, all including their characteristic dendritic profiles of both basal and apical dendrites. In the hippocampus, a variety of pyramidal, granular and non-pyramidal celltypes were stained in various hippocampal cell layers, in the dentate hilus and in stratum oriens of cornu ammonis. Moreover, positively reacting cells occurred in central and lateral amygdala, all parts of the basal ganglia and ventral pallidum. The thalamus was very richly provided with labeled neurons in several nuclei but notably numerous in the ventrolateral, anteroventral and geniculate nuclei. In cortex and hippocampus also some staining of astrocytes occurred. Electron microscopic study of the intracellular distribution of M35 immunoreactivity in all cases showed dense precipitates in the soma cytoplasm in close association with the golgi apparatus, but conspicuous absence near the endoplasmic reticulum. Immunoprecipitate can be followed within the dendritic tree along the microtubular transport system, up to proximal and distal postsynaptic membrane positions, apposing non labeled presynaptic endings. Muscarinic receptor subtype recognition by M35 will be discussed by comparing M35 distribution with cholinergic innervation patterns, muscarinic receptor ligand binding studies and localization of muscarinic receptor subtype mRNAs.     

Demonstration of muscarinic acetylcholine receptor-like immunoreactivity in the rat forebrain and upper brainstem

The distribution of muscarinic acetylcholine receptor protein (mAChR) in the rat forebrain and upper brainstem was described by using a monoclonal antibody (M35) raised against mAChR purified from bovine forebrain homogenates. A method is investigated for light microscopic (LM) and electronmicroscopic (EM) immunocytochemical visualization of reactivity to mAChR-proteins. Putative cholinoceptive neurons including their dendrites were found immunoreactive in the cortical mantle, hippocampus, basal ganglia, amygdala, thalamus and several midbrain regions. In the neocortex, immunoprecipitate with M35 was mainly present in layer 5 pyramidal cells, some layer 3 pyramidal neurons and layer 2 stellate cells, all including their characteristic dendritic profiles of both basal and apical dendrites. In the hippocampus, a variety of pyramidal, granular and non-pyramidal celltypes were stained in various hippocampal cell layers, in the dentate hilus and in stratum oriens of cornu ammonis. Moreover, positively reacting cells occurred in central and lateral amygdala, all parts of the basal ganglia and ventral pallidum. The thalamus was very richly provided with labeled neurons in several nuclei but notably numerous in the ventrolateral, anteroventral and geniculate nuclei. In cortex and hippocampus also some staining of astrocytes occurred. Electron microscopic study of the intracellular distribution of M35 immunoreactivity in all cases showed dense precipitates in the soma cytoplasm in close association with the golgi apparatus, but conspicuous absence near the endoplasmic reticulum. Immunoprecipitate can be followed within the dendritic tree along the microtubular transport system, up to proximal and distal postsynaptic membrane positions, apposing non labeled presynaptic endings. Muscarinic receptor subtype recognition by M35 will be discussed by comparing M35 distribution with cholinergic innervation patterns, muscarinic receptor ligand binding studies and localization of muscarinic receptor subtype mRNAs.     

Parvalbumin-immunoreactive structures in the hippocampus of the human adult

Summary Parvalbumin-immunoreactive structures in the fascia dentata and Ammon's horn of the adult human brain were studied using the avidin-biotin-peroxidase technique. Thin fibres (probably axons) were found to form dense networks throughout the cellular layers. Parvalbumin immunoreactivity is observed in even distal portions of nerve cell processes. The excellent quality of the immunoreaction renders the distinction of a large number of possible neuronal types. All parvalbumin-immunoreactive neurons belong to the class of non-granule cells in the fascia dentata and non-pyramidal neurons in Ammon's horn. The fascia dentata harbours four types of neurons in the molecular layer, one type within the granule cell layer and four types in the plexi-form layer. The frequently described basket cells are contained in the group of immunoreactive non-granule cells in the plexiform layer. In field CA4 two neuronal types can be distinguished. Field CA3 reveals a slender cell type in the stratum radiatum, three types in the pyramidal cell layer and three types in the stratum oriens. In field CA2 three neuronal types can be differentiated in the stratum pyramidale. The extended field CA1 is endowed with two types of nerve cells within the stratum moleculare, two types in the stratum radiatum, five neuronal types in the stratum pyramidale, and one spindle-shaped type in the stratum oriens. The morphological features of parvalbumin-immunoreactive neuronal types in the adult human brain are compared with those found in Golgi-studies of mostly young animals or in labelling experiments. This study serves as a basis for further analyzes involving specific diseases such as Alzheimer's disease or epilepsy, where it needs to be clarified to which extent certain neuronal types are afflicted.     

Distribution of GAD67-expressing neurons and morphological changes in hippocampal structures during pubertal period after acute perinatal hypoxia in rats

We investigated structural changes in the Wistar rat hippocampal CA1 field and fascia dentate during the pubertal period (on P60) after perinatal hypoxic exposure as well as the distribution of GAD67-expressing neurons in these structures. It was established that in the granular layer of the fascia dentata and in the CA1 field acute perinatal hypoxia leads to irreversible homotypic abnormalities as expressed in the reduced number of neurons and their rows as well as injury of a considerable portion of cells, which exhibit the signs of chromatolysis and vacuolization of the cytoplasm. Both in experimental and control animals, GAD67-expressing neurons in the fascia dentata are scattered diffusely and share approximately the same size of their populations. In the CA1 field, immunoreactive neurons lie in the lower rows of the pyramidal layer, while neurons in the upper layers exhibit no immunolabeling and have less synaptic structures in experimental animals than in control. We suggest that neurons in the hippocampal structures are involved in the regulation of functions and formation of prenatal pathology.     

Localization of biogenic amines and monoamine oxidase in the rabbit hippocampus

Comparison of the localization of monoamines and monoamine oxidase in the rabbit hippocampus shows that most pyramidal and all granular neurons contain no monoamines. Up to 1% of pyramidal and about 3% of polymorphic neurons are noradrenergic, and some of the latter are basket cells. Their terminals, containing both noradrenalin and monoamine oxidase, are in contact with the bodies and processes of the pyramidal and granular neurons. Single serotoninergic polymorphic neurons are found in sectors H₁ and H₂ of the cornu ammonis and in sector H₅ of the fascia dentata of the hippocampus; they have few terminals. Noradrenergic afferents enter the cornu ammonis in the external bundle of the alveus and in the septal tract, which runs along the inner surface of the fimbria. Noradrenergic terminal plexuses surround the bodies of the pyramidal cells and are concentrated at the level of the apical dendrites of the pyramids and at the base of the granular neurons of the fascia dentata. Convergence of noradrenergic and serotoninergic terminals is found on some pyramidal, granular, and polymorphic neurons. The high concentration of serotonin in the hippocampus despite the minimal number of serotoninergic neurons can be explained by the large number of serotonin-containing stellate cells of nonneural nature. They are localized on groups of pyramidal neurons in sectors H₁ and H₂ and also on blood vessels. Individual variations are found in the number of serotonin-containing neurons in the hippocampus and in the number and distribution of serotonin-containing stellate cells.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 1, pp. 40–46, January–February, 1973.     

TOPOGRAPHICAL AND SUBCELLULAR LOCALIZATION OF CHOLINE ACETYLTRANSFERASE IN RAT HIPPOCAMPAL REGION

—Choline acetyltransferase (ChAc) was localized in discrete layers in hippocampus regio superior and in area dentata. The highest activity in hippocampus was found in a narrow infrapyramidal zone, but high activities were also observed in the rest of stratum oriens and in stratum pyramidale. In area dentata the highest activities were found in a narrow supragranular zone and in hilus fasciae dentatae. The localization corresponded very closely to that of acetylcholinesterase. The main part of ChAc activity was confined to the synaptosome fraction. The results are compatible with the view that pyramidal and granular cells are excited by cholinergic boutons, mainly located on the basal or apical dendrites near the somata.     

Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult

In the present study, we focused upon expression and changes of endogenous insulin-like growth factor-1 (IGF-1) in the hippocampus of the Mongolian gerbil after ischemic insult. In sham-operated animals, IGF-1 immunoreactivity was absent from the hippocampus. IGF-1-immunoreactive (IR) neurons were detected at 12 h and 1 day after ischemic insult. In the hippocampal CA1 area, the IGF-IR neurons were non-pyramidal cells (GABAergic neurons). In the hippocampal CA2/3 areas, the IGF-1-IR neurons were pyramidal and non-pyramidal cells, and in the dentate gyrus the IGF-1-IR neurons were hilar neurons. Four days after ischemia-reperfusion, IGF-1 immunoreactivity disappeared from neurons, and significantly increased in astrocytes and microglia. These results suggest that the induction of IGF-1 in the CA1 area during the early stage (12-24 h after ischemic insult) is associated with the relative vulnerabilities of pyramidal glutamatergic neurons and non-pyramidal GABAergic neurons. The later increase (4 days after ischemic insult) of IGF-1 expression and protein content was found to promote the activities of astrocytes and microglia. These increases of IGF-1 in astrocytes and in microglia are associated with mechanisms that compensate for the effects of delayed neuronal death.     

Somatostatin-like immunoreactivity in non-pyramidal neurons of the human entorhinal region

Summary The distribution of somatostatin-immunoreactive cells and processes throughout the human entorhinal region and subjacent white matter was examined either by the unlabelled antibody-enzyme method or by the avidin-biotin method. The brain slices were obtained at autopsy with a short post-mortem delay. The majority of somatostatin immunoreactive nerve cells was found in the inner principal layer and subjacent white matter. In addition, individually scattered immunoreactive neurons were observed in both the outer principal layer and lamina dissecans. The immunoreactive perikarya varied in shape and ranged in size from 10 to 30 m. Without exception the neurons could be classified as belonging to the group of non-pyramidal neurons. Each neuron gave rise to a few thick dendrites and a thin axon with a beaded appearance. In the adult human brain, the pattern formed by lipofuscin granules deposited in the nerve cells can be considered characteristic for the type of the neuron. Therefore, immunoreactive perikarya were documented, destained of chromogen and restained to demonstrate lipofuscin pigment and basophilic substance. It became evident from these studies that the previously immunoreactive cells were characterized by a large rounded and eccentrically located nucleus, sparse basophilic substance and, in most cases, a lack of lipofuscin granules. A few of the immunoreactive cells were laden with coarse pigment granules. The findings permit classification of entorhinal somatostatin-immunoreactive neurons as either non-pigmented or pigment-laden non-pyramidal neurons.Dedicated to Prof. Dr. J. Lang, Würzburg, on the occasion of his 65th birthdayA portion of the results has been presented at the annual meeting of the European Neuroscience Association 1986 in Marseille, France (Friederich-Ecsy et al. 1986)     

Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro

Several studies have suggested that the development of cholinergic properties in cranial parasympathetic neurons is determined by these cells' axial level of origin in the neural crest. All cranial parasympathetic neurons normally derive from cranial neural crest. Trunk neural crest cells give rise to sympathetic neurons, most of which are noradrenergic. To determine if there is an intrinsic difference in the ability of cranial and trunk neural crest cells to form cholinergic neurons, we have compared the development of choline acetyltransferase (ChAT)-immunoreactive cells in explants of quail cranial and trunk neural crest in vitro. Both cranial and trunk neural crest explants gave rise to ChAT-immunoreactive cells in vitro. In both types of cultures, some of the ChAT-positive cells also expressed immunoreactivity for the catecholamine synthetic enzyme tyrosine hydroxylase. However, several differences were seen between cranial and trunk cultures. First, ChAT-immunoreactive cells appeared two days earlier in cranial than in trunk cultures. Second, cranial cultures contained a higher proportion of ChAT-immunoreactive cells. Finally, a subpopulation of the ChAT-immunoreactive cells in cranial cultures exhibited neuronal traits, including neurofilament immunoreactivity. In contrast, neurofilament-immunoreactive cells were not seen in trunk cultures. These results suggest that premigratory cranial and trunk neural crest cells differ in their ability to form cholinergic neurons.     

Lhx8 promote differentiation of hippocampal neural stem/progenitor cells into cholinergic neurons in vitro

Lhx8, also named L3, is a recently identified member of the LIM homeobox gene family. Previously, we found acetylcholinesterase (AChE)-positive cells in fimbria?Cfornix (FF) transected rat hippocampal subgranular zone (SGZ). In the present study, we detected choline acetyltransferase (ChAT)-positive cholinergic cells in hippocampal SGZ after FF transaction, and these ChAT-positive cells were double labeled by Lhx8. Then we overexpressed Lhx8 during neural differentiation of hippocampal neural stem/progenitor cells on adherent conditions using lentivirus Lenti6.3-Lhx8. The result indicated that overexpression of Lhx8 did not affect the proportion of MAP2-positive neurons, but increased the proportion of ChAT-positive cells in vitro. These results suggested that FF-transected hippocampal niche promoted the ChAT/Lhx8-positive cholinergic neurons generation in rodent hippocampus, and Lhx8 was not associated with the MAP2-positive neurons differentiation on adherent conditions, but played a role in the specification of cholinergic neurons derived from hippocampal neural stem/progenitor cells in vitro.     

Septal afferents to the area dentata terminate on vasoactive intestinal polypeptide (VIP)-like immunoreactive,non-pyramidal neurons

Summary Thermic lesions in the medial septum of adult rats result in dark degeneration of terminal boutons in the stratum moleculare and hilus of the area dentata. While most of the degenerating terminals are in synaptic contact with non-reactive cells, part of them end on dendrites of VIP-like immunoreactive neurons.     

Structural abnormalities of hippocampus in 101/HY mice

We studied cytoarchitectonics of the hippocampus in 101/HY and CBA mice on brain sections stained after Nissl and Timm. In CBA mice, the structure of hippocampus was normal. In 10/HY mice, stratum pyramidale in field CA3 was "splitted" and the density of pyramidal neurons was decreased. Abnormalities were also found in the zone of suprapyramidal projections of mossy fibers (sp-ME), i.e., terminals of axons of the fascia dentata granular cells on the apical dendrites of pyramids. If in CBA mice the sp-MF zone was normal, i.e., looked like a vast compact formation or dense ordered bundle, in 101/HY mice, the sp-MF zone represented a group of scattered, diffuse, and interrupted bundles of varying length, some of which were incorporated in stratum pyramidale. Possible causes of the described morphological abnormalities are discussed, as well as their relation to specific features of biology, behavior, and neurological status of 101/HY mice.     

GABAergic cell types in the lizard hippocampus.

The neurochemical classification of GABAergic cells in the lizard hippocampus resulted in a further division into four major, non-overlapping subtypes. Each GABAergic cell subtype displays specific targets on the principal hippocampal neurons. The synaptic targets of the GABA/neuropeptide subtype are the distal apical dendrites of principal neurons. Calretinin- and parvalbumin-containing GABAergic cells synapse on the cell body and proximal dendrites of principal cells. Calbindin is expressed in a distinct group of interneurons, the synapses of which are directed to the dendrites of principal neurons. Finally, another subtype displays NADPH-diaphorase activity, but its synaptic target has not been established.     

The features of the hippocampal structure as a neuroanatomical basis for differences in behavior

Neuromorphological and behavioural studies have been made on several strains of mice (C57B1, DBA/2, SEC) and on the spiny mouse Acomys cahirinus. It was shown that animals with different genotypes differ by the size of fascia dentata and by the extension of the pyramidal layer in CA3 field of the hippocamp. Animals with a higher learning capacity exhibited smaller layer of granular cells in the fascia dentata, which may be due to a lower density of neurones in this region. Terminals of the mossy fibers--the axons of granular cells--were found mainly on the apical dendrites of the pyramidal cells in CA3 field. On the contrary, in animals with lower capacities to learning, mossy fiber terminals were observed mainly on the basal dendrites of the pyramidal cells, the extent of the granular layer in these animals being significantly larger.     

Synaptic Contacts of Neurons of Fascia Dentata Transplants with Nonspecific Targets in Neocortex of Recipients

We carried out an electron microscopy study of possible synaptic contacts of the neurons of intracortical transplants of the rat brain fascia dentata with targets in the recipient somatosensory cortex. The axons of fascia dentata granular cell and their synaptic terminals could be easily identified in the neocortex due to their distinct morphological features (mossy fibers), although the fascia dentate cells normally do not interact with the neocortex. Thin nonmyelenized mossy fibers were found in both an intermediate zone between the transplant and brain and in the adjacent brain. Their presynaptic buds, like in situ, had large size and formed characteristic terminal, intraterminal, and en passant multiple synaptic contacts and desmosome-like junctions. The aberrant nerve fibers used perykaryons, dendrites of varying diameter, and dendrite spikes of the somatosensory cortex pyramidal neurons as postsynaptic targets in the neocortex. In addition to vacant spaces that appeared in the brain as a result of transplantation, the ingrowing axons induced the formation of additional contact sites: deep invaginations of the plasmalemma of perykaryons, somatic spikes, terminal branchings of dendrites, and dendritic outgrowths of complex branched shape. These aberrant contacts were characterized by the presence of polyribosomes, endoplasmic reticulum cisternae, and mitochondria in the postsynaptic loci. Osmiophility and extension of desmosome-like junctions were also enhanced in such synapses. Thus, it was shown that mossy fibers ingrowing in the recipient neocortex were capable of forming cell-to-cell contacts with signs of functional synapses to atypical cell targets.     

Effects of estrogens on cholinergic neurons in the rat basal nucleus

Estrogen replacement in postmenopausal women may help prevent or delay development of Alzheimer's disease. Because loss of basal forebrain cholinergic neurons with reductions in choline acetyltransferase (ChAT) concentration are associated with Alzheimer's disease, we investigated the effect of estradiol (E(2)) and J 861, a non-feminizing estrogen, on cholinergic neurons in the basal forebrain. Ovariectomized rats received E(2), J 861 or vehicle, and basal forebrain sections through the substantia innominata, medial septum, and nucleus of the diagonal band were immunostained for ChAT. ChAT-immunoreactive cells in the basal forebrain were significantly reduced in the ovariectomized rats compared to intact rats, but those ovariectomized rats receiving estrogen replacement with E(2) and J 861 had near normal levels of ChAT-positive neurons. While retrograde tracing experiments with fluorogold injected into the prefrontal cortex showed no significant differences in the number of fluorogold-labeled cells among the groups, ChAT-immunoreactive cells and double-labeled cells were significantly lower in OVX rats than in intact and E(2) rats. Some substantia innominata cells in the J 861 rats were ChAT/estrogen receptor alpha-positive. These results suggest that E(2) and J 861 have positive effects on cholinergic neurons that project from the basal nucleus to the forebrain cortex.     

The establishment of polarity by hippocampal neurons: the relationship between the stage of a cell's development in situ and its subsequent development in culture

Neurons removed from the embryonic hippocampus and placed into culture develop structurally and functionally distinct axonal and dendritic processes. The central issue addressed in this study concerns the extent to which the sequence of events which results in the differentiation of neurites by hippocampal neurons in culture is influenced by the cell's state of development in situ. [3H]thymidine was administered to pregnant rats either on Embryonic Day 15 (E15) or on E18.5 to label hippocampal neurons at known stages of their development. All fetuses were sacrificed on E19. Some of the fetal brains were sectioned and examined by autoradiography to determine the location of labeled cells in the hippocampus. The remaining brains were used to prepare hippocampal cell cultures. Neurons labeled at E18.5 remained confined to the ventricular zone at E19. Those labeled at E15 had completed their migration to the cortical plate. Other data suggest that the former cells had not yet initiated process outgrowth, while the latter cells had begun to elaborate both axons and dendrites. When introduced into culture, both populations of cells developed axons and dendrites and both compartmentalized MAP2 to the dendritic domain. Moreover, despite marked differences in their developmental state at the time of introduction into culture, both underwent the same sequence of developmental events leading to axonal and dendritic development. In a few cases cells that incorporated [3H]thymidine in situ at E18.5 apparently underwent mitosis in culture. These neurons also developed axons and dendrites appropriately. These results indicate that hippocampal neurons become polarized in culture, even if they have never developed axons or dendrites in situ, and do so as efficiently as cells that have become polarized before being placed into culture. Moreover, they indicate that the same sequence of events leading to the establishment of polarity occurs for hippocampal neurons with different developmental histories prior to culturing.