Neuronal Cell Density Research Articles

Clinical correlations with hippocampal atrophy

There is now a consensus that magnetic resonance imaging (MRI) is a sensitive and specific indicator of mesial temporal sclerosis (MTS) in patients with partial epilepsy. MTS is the most common pathological finding underlying the epileptogenic zone in patients undergoing temporal lobe surgery for medically refractory partial seizures. MRI-based hippocampal volumetric studies (i.e., quantitative MRI), has been shown to provide objective evidence for hippocampal atrophy in patients with MTS. The hippocampal volume in the epileptic temporal lobe has correlated with the neuronal cell densities in selected hippocampal subfields. A history of febrile seizures in childhood and age of unprovoked seizure onset have been associated with MRI-based hippocampal volumetry. There is conflicting evidence regarding the relationship between the duration of the seizure disorder and volumetry. Quantitative MRI has compared favorably to other noninvasive techniques (e.g., scalp-recorded EEG), in indicating the diagnosis of medical temporal lobe epilepsy (MTLE). MRI-identified hippocampal atrophy has also been a favorable prognostic indicator of seizure outcome after temporal lobe surgery. The presence of hippocampal atrophy appears to serve an in vivo surrogate for the presence of MTS.

Chronic MPTP treatment reproduces in baboons the differential vulnerability of mesencephalic dopaminergic neurons observed in parkinson's disease

Chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to baboons was shown previously to result in a motor syndrome and a pattern of striatal dopaminergic fibre loss similar to those observed in idiopathic Parkinson's disease. In the present study, tyrosine hydroxylase-immunoreactive neurons were quantified in the mesencephalon of control ( n = 4) and chronically MPTPtreated ( n = 3) baboons. MPTP induced a significant reduction in neuronal cell density in the substantia nigra (63.8% reduction) and the ventral tegmental area (53.1%). Within the substantia nigra, obvious mediolateral and dorsoventral gradients of neuronal cell loss were observed. First, the pars lateralis was more affected than the lateral divisions of the pars compacta (89.6% vs 73.8% cell loss), which in turn were more depleted than the medial divisions (60.1% reduction). Second, the ventral regions of the pars compacta were more degenerated than the dorsal parts (82.4 vs 51.5% decrease). This regional pattern is strikingly similar to that observed in Parkinson's disease and indicates that two subpopulations of dopaminergic neurons are distinguishable on the basis of their differential vulnerability to MPTP. Finally, the present study confirms that chronic mitochondrial complex I inhibition using MPTP in primates is sufficient to reproduce the typical dopaminergic cell loss and striatal fibre depletion observed in Parkinson's disease.

Isolation and Characterization of a Mouse Homolog of the Drosophila Segment Polarity Gene dishevelled

In the Drosophila embryo dishevelled (dsh) function is required by target cells in order to respond to wingless (wg, the homolog of Wnt-1), demonstrating a role for dsh in Wnt signal transduction. We have isolated a mouse homolog of the Drosophila dsh segment polarity gene. The 695-amino-acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. Similarity searches of protein and DNA data bases revealed that Dvl-1 encodes an otherwise novel polypeptide. While no functional motifs were identified, one region of Dvl-1 was found to be similar to a domain of discs large-1 (dlg), a Drosophila tumor suppressor gene. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with brain and testis exhibiting the highest levels. The majority of Dvl-1 expression in the adult cerebellum is in the granular cell layer, similar to the pattern seen for engrailed-2 (En-2) . Throughout postnatal development of the brain Dvl-1 is highly expressed in areas of high neuronal cell density.

Entorhinal-hippocampal interactions in medial temporal lobe epilepsy.

Experimental studies suggest important interactions between hippocampus and entorhinal cerebral cortex in generation of temporal lobe seizure activity. We studied electrical expression of spontaneous temporal lobe ictal activity in hippocampus and entorhinal cortex in 9 medically refractory epileptic patients who had intracranial depth and subdural electrodes implanted during surgical evaluation. All 9 patients subsequently underwent anteromedial temporal lobectomy with hippocampectomy, all had > 50% decrease in neuronal cell density in hippocampal CA1 and CA3, and all had good to excellent seizure outcome after operation. Two to 10 spontaneous seizures were analyzed per patient (total 41 seizures). Nine patients had variable onset of seizure activity recorded in hippocampus, entorhinal cortex, or both simultaneously. Low-voltage fast activity was observed in either location and varied among seizures in an individual patient. Periodic preictal spikes, when present, were often synchronous in both locations, but were noted independently only in hippocampus. Our data suggest that preictal spikes and low-voltage fast seizure discharges have anatomically distinct origins, and that some syndromes of medial temporal lobe epilepsy involve interactions between entorhinal and hippocampal regions that act together to produce and propagate the seizures in such patients.

Glia‐derived nexin/protease nexin‐1 is expressed by a subset of neurons in the rat brain

Glia-derived nexin/protease nexin-1 (GDN/PN-1) is a serine protease inhibitor that is secreted by glial cells and fibroblasts in culture. In the adult mammalian nervous system it has been shown to be expressed in the olfactory system and by some glial cells in response to neuronal injury. In situ hybridization and immunocytochemical studies were performed to identify the structures expressing GDN/PN-1 in the developing and adult rat brain. In contrast to a transient widespread expression during pre- and postnatal development, some brain structures constitutively express GDN/PN-1. These include the olfactory nerve layer of the olfactory bulb, basal forebrain, striatum, pyramidal neurons of layer V in the cortex, thalamic nuclei, pars compacta of the substantia nigra, inferior and superior colliculi, and deep cerebellar nuclei. All of these parts, excluding the olfactory nerve layer, are characterized by a high neuronal cell density. Neurons in these regions were immunoreactive for GDN/PN-1. Furthermore GDN/PN-1 expression in cell lines showed that the active protein was synthesized and secreted from B104 but not from NB2a neuroblastoma cells. Although GDN/PN-1 has only been reported to be synthesized by glia, the results presented here demonstrate that in addition, a subset of neurons express this protease inhibitor.

Diurnal and seasonal rhythms of neuronal activity in the suprachiasmatic nucleus of humans.

The mammalian suprachiasmatic nucleus (SCN) is considered to be a critical component of a neural system implicated in the temporal organization of a wide variety of biological processes. Since the environmental light-dark cycle is the main zeitgeber for many of these rhythms, photic information may have a synchronizing effect on the endogenous clock in the SCN by inducing periodic changes in the activity of certain groups of neurons. The present study was conducted to investigate whether the daily light-dark cycle as well as seasonal variations in photoperiod would affect the vasopressin cell population of the human SCN. To that end, the brains of 30 young human subjects (ranging in age from 6 to 47 years) were investigated. We found that the subdivision of the human SCN that contains vasopressin-producing neurons fluctuated significantly over the 24-hr period. The volume of the vasopressin cell population was, on average, 1.4 times as large during the daytime (1000-1800 hr) as during the nighttime (2200-0600 hr), and contained 1.8 times as many vasopressin-immunoreactive neurons. Peak values in both vasopressin volume and vasopressin cell number were observed in the early morning (0600-1000 hr). In general, the SCN contained fewer vasopressin-immunoreactive neurons during the night than during any other period of the natural light-dark cycle. In addition to the diurnal cycle of the SCN, a marked seasonal rhythm was observed. The volume of the vasopressin cell population was, on average, 2.4 times as large in the autumn as in the summer, and contained 3 times as many vasopressin-immunoreactive neurons. In general, the annual cycle of the human SCN showed a nonsinusoidal pattern with a maximum in early autumn, a lower plateau in winter, and a deep trough in late spring and early summer. In contrast with the periodic fluctuations in the number of vasopressin-immunoreactive neurons in the SCN, no significant diurnal or seasonal variations could be detected in the numerical cell density or cell nuclear diameter of vasopressin neurons. In conclusion, the findings indicate that the synthesizing activity of the vasopressin neurons of the human SCN exhibits a diurnal as well as a seasonal rhythm, and that the temporal organization of these processes becomes disturbed later in life.

Intrathecal injection of lysine acetylsalicylic acid in the rat: a neurotoxicological study

Lysine acetylsalicylic acid has been reported to induce analgesic effects in humans after intrathecal (i.t.) injection. Before conducting further studies in humans with this drug, it is important to evaluate potential toxicological effects on the spinal cord in animals. In the present study the effects of chronic intrathecal administration of provocative doses of lysine acetylsalicylic acid (L-ASA) on the rat spinal cord were evaluated using light and electron microscopy and a quantitative morphometric method. We also investigated the effects of single doses of the drug on the spinal cord blood flow (SCBF) using the laser-Doppler flowmetry technique. No histopathological changes or differences in number or density of neuronal cells could be seen after chronic administration of L-ASA as compared to controls. The SCBF decreased immediately after i.t. injection of a large dose (4 mg) of L-ASA and returned to predrug levels within 10 min. At the end of the experiment metabolic acidosis was detected, indicating a systemic effect of acetylsalicylic acid. It is concluded that no neurotoxic effects on the spinal cord were seen after chronic i.t. injection of L-ASA. From a neurotoxicological point of view, our findings do not contraindicate the spinal use of L-ASA in humans.

A neurotoxicologic evaluation of the spinal cord after chronic intrathecal injection of R-phenylisopropyl adenosine (R-PIA) in the rat.

Studies in animals have shown that the adenosine receptor agonist R-phenylisopropyl adenosine (R-PIA) induces antinociceptive effects after intrathecal administration. Before such a potentially antinociceptive drug could be considered for intrathecal injection in humans, a neurotoxicologic examination of the spinal cord must be performed in animal models. Rats were injected once every day for 14 consecutive days with R-PIA or saline (controls). The number and density of neuronal cells were calculated by using light microscopy, and further examined with electron microscopy. The "disector method," which is an unbiased stereologic estimator of cell number and mean cell volume, was used for quantitative morphometric analyses. With this technique no significant changes could be seen in rats that had received R-PIA as compared to control rats. We conclude that no significant histologic changes could be detected after chronic intrathecal administration of R-PIA.

A histometrical study on the globus pallidus in Huntington's disease

Pathological change of the globus pallidus (GP) in 6 cases of Huntington's disease (HD) was examined histometrically by comparison with 10 normal control cases. All but 1 case of HD were in late stages of the disease. Total neuronal count, area of GP, and neuronal cell density were measured in 5 selected regions of coronal sections taken along the antero-posterior axis. Contrary to the findings of previous reports, no neuronal depletion was recognized in HD in any region despite marked atrophy of tissue bulk. The atrophy was more severe in the external segment (GP e) than in the internal segment (GP i). Reactive astrocytosis and fibrillary gliosis were observed in the atrophic lesions. These results indicate that atrophy of the GP can be attributed to striato-pallidal fiber loss and not to neuronal depletion even in the late stages. These findings support the hypothesis that loss of striato-GP e fibers plays the most important role in choreic movements in HD. It remains to be determined whether the pallidal neurons are also preserved in the end stage of the disease.

Dorsal motor nucleus of the vagus nerve: A cyto‐ and chemoarchitectonic study in the human

In order to investigate the topography and subdivisions of the human dorsal motor nucleus of the vagus nerve (10), studies were conducted using cyto- and chemoarchitectonic (acetylcholinesterase and substance P-like immunoreactivity) and computer reconstruction techniques. The six brainstems examined were obtained within 17 hours postmortem from adults with no known neurological disorders. Serial sections cut in transverse, sagittal, and coronal planes were stained with cresyl violet, or tested for acetylcholinesterase or substance P. The neurons of the 10 (16,826 +/- 967) displayed cyto- and chemoarchitectonic heterogeneity and could be classified into six types. Types I-V consist of presumed vagal motor neurons (13,802 +/- 844), while the remaining type (Type VI) consisted of presumed interneurons (3,024 +/- 769). The 10 was subdivided into nine subnuclei grouped regionally into rostral, intermediate, and caudal divisions on the basis of neuronal morphology, cell density, and differential AChE and substance P reactivities. The rostral division contains the dorsorostral (DoR) and the ventrorostral (VeR) subnuclei; the intermediate division contains the rostrointermediate (RoI), dorsointermediate (DoI), centrointermediate (CeI), ventrointermediate (VeI), and caudointermediate (CaI) subnuclei; the caudal division (Ca) is not subdivided. Morphologically, small round or oval cells populate the VeR and VeI. Medium-sized oval cells occur in the DoR, CeI, and Ca, while medium-sized fusiform and multipolar cells are the main features of CaI. Large triangular cells appear mainly in DoI. Glial cells show the highest predilection for CeI, lowest densities in DoI and medial fringe subnucleus (MeF), and intermediate densities in the remaining six subnuclei. VeI showed the strongest AChE reactivity. Although the cell bodies of VeR and DoI are AChE positive, the neuropil (background) is weakly stained. Densely distributed fine granular substance P-like immunoreactivity occurs throughout the entire nucleus, while the intermediate and caudal divisions contain substance P-like-immunoreactive neurons. Three-dimensional computer reconstructions afforded an appreciation of the distinctiveness of the intermediate division (a division that contains the majority of cells) and the caudal division, which displays the lowest density of presumed vagal motoneurons. It is possible that the subnuclei identified herein form functional units innervating specific organs.

Formation and maturation of synapses in primary cultures of rat cerebral cortical cells: an electron microscopic study

Cerebral cortical cells from fetal rats (18 days) were cultured for 3, 7, 14, 21, 28, 35 days in vitro (DIV) and the development of synapses was examined morphologically by electron microscopy. At 3 DIV, no synapses could be recognized. An immature type of synapse was found at 7 DIV which thereafter developed morphologically. The length of the synaptic contact zone (SCZ) increased with DIV from 271 ± 11.4 nm (mean ± SEM) at 7 DIV to 410 ± 11.4 nm at 35 DIV. The number of synaptic vesicles per terminal also increased with DIV: 10.0 ± 1.2 at 7 DIV, 35.7 ± 3.4 at 21 DIV, and 53.3 ± 4.5 at 35 DIV. The time course of numerical density of synapses was examined quantitatively by electron microscopy. Synaptic density was very low at 7 DIV. It was significantly increased at 14 DIV and thereafter showed variable changes. Four culture series showed decreases after 14 DIV, but one series showed a further increase at 21 DIV followed by a decrease at 28 DIV. The mean density of synapses at each DIV was as follows: 1780 ± 86 10 6 μ m 3 at 7 DIV, 4244 ± 595 10 6 μ m 3 at 14 DIV, 2285 ± 674 10 6 μ m 3 at 21 DIV, 2552 ± 646 10 6 μ m 3 at 28 DIV, and 2080 ± 532 10 6 μ m 3 at 35 DIV. Neuronal cell density was counted in each culture to calculate the relative number of synapses per neuron. The cell density decreased with age from 301 ± 51 10 6 μ m 3 at 7 DIV to 39 ± 9 10 6 μ m 3 at 35 DIV. The number of synapse per neuron increased with DIV to the end of the 4th week (from 64.1 ± 9.8 at 7 DIV to 1130.1 ± 254.3 at 28 DIV). The time course of synapse formation in culture resembled that in the cerebral cortex in vivo.

O-180 - Temporal pattern of changes in extracellular acetylcholine levels and neuronal cell density in gerbil hippocampus following transient ischemia.

O-180 - Temporal pattern of changes in extracellular acetylcholine levels and neuronal cell density in gerbil hippocampus following transient ischemia.

Regional differentiation of cell densities in the left and right hippocampi of epileptic patients.

Neuronal cell densities in surgically removed left or right hippocampal tissue of epileptic patients suffering from temporal lobe epilepsy were determined in CA1, CA4, and the dentate gyrus (DG). Mean densities showed no statistically significant left-right asymmetry in CA1 and the DG, consistent with findings by others. Mean densities did show previously unreported asymmetry in CA4; densities were significantly lower in the right CA4 than in the left. Another new finding is an asymmetry in regional intercorrelations: positive and significant correlations among the three subfields were obtained only in the left hippocampus. In addition, we confirmed previous findings of positive correlation between neuronal densities in CA1 and age of onset of habitual epilepsy, on either side. Taken together, the results suggest subtle left-right asymmetries in the vulnerability of the hippocampi to epilepsy-associated damage and/or higher neuronal connectivity or interdependence on the left than on the right.

Gliosis and ganglion cell death in the developing cat retina during hydrocephalus and after decompression

Even after surgical decompression, infantile hydrocephalus often results in permanent neurological symptoms, including visual deficits. However, little is known about the cellular changes that may be responsible for these effects. The present study was designed to analyze the retinae of normal, midly hydrocephalic, severely hydrocephalic and surgically decompressed kittens to determine if changes occur in the density and size of retinal ganglion cells. Hydrocephalus was induced in 10 day old kittens by intra-cisternal injection of kaolin. Kittens were allowed to survive from 7 to 28 days after injection. Animals that were decompressed received ventriculoperitoneal shunts 10–15 days after the induction of hydrocephalus and were sacrificed 10–14 days after shunt placement. The density and area of neuronal and glial cells were determined within a sample area in peripheral nasal retina. Total cell density was significantly increased in midly and severely hydrocephalic animals but returned to normal following decompression. This change represents a significant increase in the glial population. In addition, there was a significant loss of ganglion cells in both the severely hydrocephalic and the shunted groups. Based on these findings, we conclude that gliosis occurs as a result of cell death in the retina following severe hydrocephalus, and decompression is unable to reverse these effects. Furthermore, gliosis occurs in mild cases of hydrocephalus, and may be an early indication that cellular degeneration will follow.

Pathology of the cerebellar dentate and interpositus nuclei in Joseph disease: a morphometric investigation

Cerebellar dentate and interpositus nuclei pathology was studied morphometrically in 3 patients with Joseph disease compared to 3 control subjects. Size of neurons, number of small neurons (cell body area: 50–199 μm 2) and large neurons (cell body area: 200 μm 2 or greater) at the rostral, medial and caudal levels, neuronal cell density, total volume of the gray bands, and total neuronal cell number were evaluated in the dentate, emboliform, and globose nuclei, using an image analyzer, after making horizontal serial 20-μm thick sections of a unilateral cerebellar hemisphere embedded in celloidin. The number of large neurons in Joseph disease was around 20% of that in the controls at each level in the individual nucleus ( P <0.05 or P <0.01). In contrast, the number of small neurons was significantly reduced only at the caudal level of the dentate nucleus ( P <0.05). The neuronal cell density was decreased within the nuclei. The total volume of the gray bands was reduced to about 70% within the nuclei ( P<0.05 or P<0.01). The total number of neurons was decreased to about a third, a half, and a third within the dentate ( P<0.001), emboliform ( P<0.01), and globose nuclei ( P<0.001), respectively. The principal pathologic change of the dentate and interpositus nuclei in Joseph disease was severe loss of neurons, with significant loss of the large neurons, indicating that Joseph disease is a type of cerebellar efferent system disorder.

Structural features of neurons in whole grafts of the rat inferior colliculus

The inferior colliculus (IC) is a midbrain structure that receives ascending auditory input from brainstem nuclei via the lateral lemniscus, sends efferent fibers to the medial geniculate body of thalamus and receives descending projections from auditory cortex. In the rat, the IC consists of dorsal and external cortices surrounding the central nucleus of IC (CNIC) which is populated by discoid and stellate neurons; the CNIC has a laminar appearance arising from organization of lemniscal fibers and processes of discoid cells. The IC of adult rats was chosen for implantation of whole grafts of E16–17 caudal tectum into unilateral lesion sites. Dendritic and somal architecture of graft neurons was examined 1 to 4.5 months following implantation using rapid Golgi, HRP and Nissl methods. The CNIC of rat is dominated by principal neurons with relatively flattened dendritic fields. In grafts of caudal tectum the most common neuron class observed possesses flattened dendritic arbors which often parallel one another. These neurons also resemble CNIC neurons of host tissue adjacent to the graft border. Spine formations appear on both proximal and distal dendrites of this neural type in both normal and implanted tissues. In addition, comparable somal features of graft neurons include ovoid or fusiform shapes with regular nuclear membranes as found in the normal colliculus. In Golgi stained material fewer stellate class neurons appear as in the normal CNIC, although stellate cell classes are more abundant in the pericentral areas of normal tissue. Both neuron populations are retrogradely labelled in graft and normal IC after HRP injection into the medial geniculate body. These features suggest that the graft core typically consists of prototypic CNIC cells. Other features of neuron and glial cell density vary in graft material which also shows a complex network of vasculature. These results demonstrate that whole grafts of caudal tectum placed into the inferior colliculus can form organized neural architecture similar to the normal CNIC. The somal, dendritic and spine features of these neurons form a potential substrate for connectional and functional properties which establish this preparation as suitable for further investigation as a model for development and recovery of function in the central auditory system.

Hypertrophy and Increased Gene Expression of Neurons Containing Neurokinin-B and Substance-P Messenger Ribonucleic Acids in the Hypothalami of Postmenopausal Women*

We have previously described hypertrophy of neurons containing estrogen receptor mRNA in the infundibular nucleus of postmenopausal women. In the present investigation we identified peptide mRNAs in the hypertrophied neurons and determined whether postmenopausal neuronal hypertrophy was accompanied by changes in gene expression. In the first study in situ hybridization was performed on sections from hypothalami of postmenopausal women (n = 3) using synthetic 35S-labeled cDNA probes complementary to mRNAs encoding estrogen receptor, substance-P (SP), neurokinin-B (NKB), POMC, cholecystokinin, dynorphin, CRF, enkephalin, galanin, neuropeptide-Y, GH-releasing hormone, and tyrosine hydroxylase. Neuronal cross-sectional areas and cell densities were measured with the aid of a computer microscope system. Neurons labeled with the NKB and SP probes were comparable in size, morphology, and distribution to the hypertrophied neurons containing estrogen receptor mRNA. In contrast, neurons labeled with other cDNA probes were sparsely distributed (CRF and dynorphin), smaller in size (neuropeptide-Y, galanin, GH-releasing hormone, enkephalin, cholecystokinin, and POMC), or located anterior to the hypertrophied population (tyrosine hydroxylase). In the second study sections from hypothalami of premenopausal (n = 3) and postmenopausal (n = 3) women were incubated with cDNA probes complementary to SP or NKB mRNAs. The mean cross-sectional areas of postmenopausal infundibular neurons containing NKB and SP mRNAs increased to 194% and 176% of premenopausal values, respectively. The autoradiographic grain densities of infundibular neurons labeled with either probe were also significantly increased in the postmenopausal group. Finally, the numbers of labeled neurons/tissue increased 6-fold (SP) and 15-fold (NKB) in the postmenopausal infundibular nucleus. These data demonstrate that human menopause is associated with marked increases in hypothalamic NKB and SP gene expression. We propose that neurons containing estrogen receptor, SP, and NKB mRNAs participate in the hypothalamic circuitry regulating estrogen negative feedback in the human.

Effect of a New Calcium Entry Blocker, NB-818, on Delayed Neuronal Death in the Ischemic Gerbil Hippocampus.

The effect of NB-818, a new dihydropyridine calcium entry blocker, on delayed neuronal death (DND) in the hippocampal CA1 subfield of gerbils after 5 minutes of forebrain ischemia induced by bilateral carotid artery occlusion was examined. Gerbils were treated intraperitoneally with NB-818 (0.1-3 mg/kg) just after release of the occlusion. Four days after the ischemia, they were fixed by perfusing 10% buffered-formalin, and the neuronal cell density (NCD, cell/mm) in the CA1 subfield was estimated under microscopy. The average NCD in the ischemic control group was 43 +/- 10.8 cells/mm, whereas NB-818 (3 mg/kg) significantly ameliorated DND with an average NCD of 143 +/- 24.2 cells/mm (P less than 0.01). In addition, NB-818 (3 mg/kg) significantly inhibited DND at 1, 2 and 4 weeks after transient ischemia: the average NCD of the NB-818 and ischemic control groups were 80 +/- 9.4 (P less than 0.01) and 43 +/- 7.7 cells/mm, 92 +/- 13.7 (P less than 0.05) and 52 +/- 9.3 cells/mm, and 57 +/- 5.0 (P less than 0.01) and 43 +/- 12.4 cells/mm, respectively. In this experiment, NB-818 exhibited a protective effect on DND in the hippocampal CA1 subfield after transient forebrain ischemia, and its effect persisted for up to 4 weeks. These findings suggest that NB-818 may be useful for clinical treatment of neurological deficit after an ischemic insult.

Size and density of glial and neuronal cells within the cerebral neocortex of various insectivorian species

Morphometric measurements were done on frontal sections through the somatosensory neocortex of various insectivorian species. All measured parameters varied with the size of animals; there was a better correlation with the ventriculartopial brain wall thickness than with the brain weight. The following rules were evaluated: with increasing brain wall thickness, 1) lamina I becomes thinner; 2) the nuclei of both neuronal and glial cells become larger; 3) the volume density of neuronal cells decreases greatly; 4) the volume density of glial cells increases slightly; and 5) as a result, the glia:neuron index increases markedly. There was no equal number of neurons under a unit surface area in the cortices of any species studied. Developmental processes that might account for the above-mentioned rules are discussed in this report.

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture: I. Conditions under which neurons form axons but not dendrites

We have examined the morphology of fetal rat sympathetic neurons grown in serum-free medium in the absence of nonneuronal cells. Because cell density can affect phenotypic expression in vitro, the morphological analysis was subdivided into the study of isolated neurons (neurons whose somata were at least 150 μm from their nearest neighbor) and of more highly aggregated neurons. When isolated neurons were injected with intracellular markers, it was found that most (79%) had a single process emanating from their somata and that this unipolar state persisted for at least 8 weeks in vitro. The processes of unipolar sympathetic neurons had the appearance of axons in that they were thin and long, had a constant diameter, and were relatively unbranched. Cytochemical methods revealed that such processes had other axonal characteristics: (1) they were more reactive with a monoclonal antibody against phosphorylated forms of the M and H neurofilament subunits than with an antibody to nonphosphorylated forms of these proteins; (2) they also reacted with antibodies to the τ microtubule-associated protein and to the phosphorylated forms of the H neurofilament subunit; and (3) they contained only small amounts of RNA as determined by [ 3H]uridine autoradiography. These data indicate that neurons which normally form dendrites in vivo need not express this capacity in vitro and that axonal and dendritic growth can be dissociated under some conditions in culture. While most isolated neurons were unipolar, neurons in regions of high neuronal cell density were usually multipolar. In addition to axons, multipolar neurons had processes with some of the characteristics expected of rudimentary dendrites: they ended locally (usually within 100 μm), were often highly branched, and reacted with an antibody to nonphosphorylated forms of the M and H neurofilament subunits. The effects of density were most prominent when neurons were within aggregates in which the somata were in close apposition. Density-dependent changes in morphology were less frequently observed when neuronal somata were separated by greater distances (30–100 μm). These data indicate that the morphology of sympathetic neurons is subject to environmental regulation and that neuron-neuron interactions can promote the extension of rudimentary dendrites in vitro.