Glial cells in the guinea pig myenteric plexus are dye coupled

Glial cells in the myenteric plexus of the guinea pig small intestine were stained intracellularly with Lucifer yellow and horseradish peroxidase. The cells were identified by both their electrophysiological characteristics and by their morphology. Injection of Lucifer yellow, which is known to cross gap junctions, resulted in the staining of many (up to about 100) glial cells. The staining pattern was comparable to the immunostaining of glia with an antiserum for S-100 protein. In contrast to Lucifer yellow, horseradish peroxidase (which does not cross these junctions), was confined to the injected cell. It is concluded that enteric glia are coupled, presumably by gap junctions. This finding indicates that in addition to structural and biochemical similarities, enteric glia may share certain physiological characteristics with central nervous system astrocytes.     

Dye-coupling between glial cells in the guinea pig neocortical slice

Physiologically identified glial cells in guinea pig neocortical slices were injected with the low molecular weight, fluorescent dye Lucifer yellow CH. The stained aggregates which resulted consisted of one brightly stained, central cell surrounded by numerous lightly stained cells. The central cell had well defined feathery processes and resembled a protoplasmic astrocyte. The surrounding cells appeared also to be glial cells but lacked sufficient detail to be further categorized. This first demonstration of dye-coupling between neocortical glial cells strongly suggests that these cells are connected together via low resistance junctions capable of passing ionic current as well as dye.     

Actions of noradrenaline on myenteric neurons in the guinea pig gastric antrum.

We used intracellular electrophysiological recording to study the actions of noradrenaline on myenteric neurons in the guinea pig gastric antrum. Noradrenaline caused a dose-dependent inhibition of the stimulus-evoked cholinergic fast excitatory postsynaptic potentials (EPSPs). Noradrenaline had no effect on the postsynaptic response to acetylcholine, suggesting a presynaptic site of action. The slow EPSP was also presynaptically inhibited by noradrenaline. In only 5% of the neurons, noradrenaline caused a postsynaptic depolarization, accompanied by increased input resistance and enhanced excitability. Studies with adrenergic antagonists and agonists revealed that the presynaptic inhibitory effect was mediated by an alpha 2-receptor, while the postsynaptic excitatory effect seemed to be mediated by an alpha 1 receptor. We conclude that noradrenaline inhibits neurotransmitter release from cholinergic and non-cholinergic nerve terminals in the myenteric plexus of the antrum and that it excites a subpopulation of antral neurons. Both mechanisms may contribute to the neurally mediated inhibitory action of noradrenaline on gastric contractility.     

The enteric nervous system in tissue culture. I. Cell types and their interactions in explants of the myenteric and submucous plexuses from guinea pig, rabbit and rat

This paper describes methods for removing the ganglionated myenteric and submucous plexuses from the mammalian gut and maintaining them as explants in tissue culture. A detailed account is given of cell types, their interactions and the development of these cultures during 5 weeks in vitro.Three major cell types were identified in the cultures: neurons, glial cells and fibroblasts. The development of the plexuses in culture was studied in detail for the myenteric plexus from the guinea pig taenia coli. It followed a characteristic pattern, in which the merging of individual ganglia into a continuous monolayer of flattened neurons was accompanied and followed by the formation of an extensive outgrowth zone of flat glial cells covered by a dense mesh of outgrowing neurites. In older cultures, neuronal migration resulted in the reformation of discrete and compact aggregates, which consisted of neurons and glial cells, and were interconnected by thick neurite bundles. This arrangement resembles in many ways the original organization of enteric nervous tissue in vivo.This is the first time the enteric ganglia have been freed from the gut wall and grown in culture as explants of nervous tissue. These preparations open many new directions for investigations of the largest and most complex division of the peripheral nervous system, including studies of the molecular nature of neuronal and glial cell surfaces,analysis of cell-cell interactions, trophic factors and developmental signals.     

Projections of submucosal neurons to the myenteric plexus of the guinea pig intestine: in vitro tracing of microcircuits by retrograde and anterograde transport

The enteric nervous system (ENS) can mediate reflex activity without input from the brain or spinal cord. The ENS thus contains intrinsic primary afferent neurons that link mucosal sensory receptors with motor neurons in the myenteric plexus. The intrinsic primary afferent neurons of the gut have not yet been identified. Although the submucosal plexus is known to innervate the mucosa, where enteric sensory receptors are located, no submucosal to myenteric projections have previously been found. In order to determine whether such projections exist, the submucosal plexus was examined following the microinjection of a retrograde tracer (Fluoro-Gold or 4-acetoamido, 4'-isothiocyanostilbene-2,2'-disulphonic acid [SITS]) into single myenteric ganglia. In addition, the myenteric plexus was studied following the iontophoretic injection of an anterograde tracer (Phaseolus vulgaris leucoagglutinin; [PHA-L]) into single submucosal ganglia. Ganglia were visualized by use of differential interference contrast optics and were injected from the beveled tip of a glass micropipette; 2.5-3.0 hours were allotted for retrograde and 20-24 hours (under culture conditions) for anterograde transport. In the myenteric plexus, a small number of the neurons of each injected ganglion were fluorescent and additional neurons in distant myenteric ganglia (predominantly orad) were also retrogradely labeled. About five to six submucosal neurons deep to but not directly underneath the injected myenteric ganglion were labeled by Fluoro-Gold or SITS and only rarely was there more than one labeled neuron in a submucosal ganglion. When control injections of Fluoro-Gold were placed into the muscle instead of a ganglion, some myenteric neurons near the injection site became labeled indicating an innervation of the circular muscle by myenteric neurons; however, there was no labeling of neurons in the submucosal plexus. Similarly, if connections between the myenteric and submucosal plexuses were severed before injecting Fluoro-Gold, no submucosal neurons were labeled. Following injection of PHA-L into a single submucosal ganglion, small-diameter axons were labeled in approximately 2 myenteric ganglia as well as in several distant submucosal ganglia (mainly anal and circumferential to the injection site). Additional labeled fibers traveled with blood vessels or surrounded mucosal crypts. It is concluded that submucosal neurons project to the myenteric plexus as well as to the mucosa and to one another. These observations are consistent with the hypothesis that at least some intrinsic enteric primary afferent neurons reside in the submucosal plexus.     

Immediate-early gene expression in the inferior mesenteric ganglion and colonic myenteric plexus of the guinea pig.

Activation of neurons in the inferior mesenteric ganglion (IMG) was assessed using c-fos, JunB, and c-Jun expression in the guinea pig IMG and colonic myenteric plexus during mechanosensory stimulation and acute colitis in normal and capsaicin-treated animals. Intracolonic saline or 2% acetic acid was administered, and mechanosensory stimulation was performed by passage of a small (0.5 cm) balloon either 4 or 24 hr later. Lower doses of capsaicin or vehicle were used to activate primary afferent fibers during balloon passage. c-Jun did not respond to any of the stimuli in the study. c-fos and JunB were absent from the IMG and myenteric plexus of untreated and saline-treated animals. Acetic acid induced acute colitis by 4 hr, which persisted for 24 hr, but c-fos was found only in enteric glia in the myenteric plexus and was absent from the IMG. Balloon passage induced c-fos and JunB in only a small subset of IMG neurons and no myenteric neurons. However, balloon passage induced c-fos and JunB in IMG neurons (notably those containing somatostatin) and the myenteric plexus of acetic acid-treated animals. After capsaicin treatment, c-fos and JunB induction by balloon passage was inhibited in the IMG, but there was enhanced c-fos expression in the myenteric plexus. c-fos and JunB induction by balloon stimulation was also mimicked by acute activation of capsaicin-sensitive nerves. These data suggest that colitis enhances reflex activity of the IMG by a mechanism that involves activation of both primary afferent fibers and the myenteric plexus.     

Release of gamma-aminobutyric acid from guinea pig myenteric plexus synaptosomes

A crude synaptosomal preparation obtained from myenteric plexus of guinea pig small intestine was used to study the release of [3H]GABA. GABA release was elevated in response to depolarizing agents when tested in normal Krebs but was depressed in a calcium-free medium. GABA was also noted to stimulate a release of [3H]ACh from the same preparation. These data support the possible role of GABA as an enteric neurotransmitter.     

Dye coupling among satellite glial cells in mammalian dorsal root ganglia

Dorsal root ganglia (DRG) are key elements in sensory signaling under physiological and pathological conditions. Little is known about electrical coupling among cells in these ganglia. In this study, we injected the fluorescent dye Lucifer yellow (LY) into single cells to examine dye coupling in DRG. We found no dye coupling between neurons or between neurons and their attendant satellite glial cells (SGCs). In mouse DRG, we observed that in 26.2% of the cases SGCs that surround a given neuron were dye coupled. In only 3.2% of the cases SGCs that make envelopes around different neurons were coupled. The data from mouse ganglia were very similar to those from rat and guinea pig DRG. The results obtained by injection of the tracer biocytin were very similar to those observed with LY. The coupling incidence within the envelopes increased 3.1-fold by high extracellular pH (8.0), but coupling between envelopes was not affected. Acidic pH (6.8) reduced the coupling. High extracellular K+ (9.4 mM) increased the coupling 2.4-fold and 4.7-fold within and between envelopes, respectively. Low extracellular Ca2+ (0.5, 1.0 mM) partly reversed the effect of high K+ on coupling. The results showed that SGCs in mammalian sensory ganglia are connected by gap junctions. This coupling is very sensitive to changes in pH, and can therefore be modulated under various physiological and pathological conditions. The dependence of the coupling on extracellular K+ and Ca2+ suggests that the permeability of gap junctions can be altered by physiological and pharmacological stimuli.     

Electrical coupling between glial cells in the rat retina.

The strength of electrical coupling between retinal glial cells was quantified with simultaneous whole-cell current-clamp recordings from astrocyte-astrocyte, astrocyte-Müller cell, and Müller cell-Müller cell pairs in the acutely isolated rat retina. Experimental results were fit and space constants determined using a resistive model of the glial cell network that assumed a homogeneous two-dimensional glial syncytium. The effective space constant (the distance from the point of stimulation to where the voltage falls to 1/e) equaled 12.9, 6.2, and 3.7 microm, respectively for astrocyte-astrocyte, astrocyte-Müller cell, and Müller cell-Müller cell coupling. The addition of 1 mM Ba(2+) had little effect on network space constants, while 0.5 mM octanol shortened the space constants to 4.7, 4.4, and 2.6 microm for the three types of coupling. For a given distance separating cell pairs, the strength of coupling showed considerable variability. This variability in coupling strength was reproduced accurately by a second resistive model of the glial cell network (incorporating discrete astrocytes spaced at varying distances from each other), demonstrating that the variability was an intrinsic property of the glial cell network. Coupling between glial cells in the retina may permit the intercellular spread of ions and small molecules, including messengers mediating Ca(2+) wave propagation, but it is too weak to carry significant K(+) spatial buffer currents.     

Short-term nerve stimulation increases enkephalin production and content in the guinea pig myenteric plexus

Methionine-enkephalin content in the guinea pig myenteric plexus was determined before and after acute, short-term electrical or chemical stimulation. Stimulation at 20 Hz for 30 s or exposure to high potassium, the calcium channel agonist, CGP28 392, or the narcotic antagonist, (-)-naloxone, resulted in a significant increase in the content of myenteric methionine-enkephalin. The increase produced by electrical stimulation is dependent upon functional sodium channels and the presence of extracellular calcium. These results indicate that tissue levels of enkephalin are not fixed but can fluctuate in response to nerve stimulation and suggest a mechanism whereby the rate of production of this opioid peptide is coupled to neuronal activity. Furthermore, the ability of (-)-naloxone but not (+)-naloxone to almost double myenteric enkephalin content suggests that the neurons in which this increase occurs are under tonic modulation (direct or indirect) by opioids.     

Projections of submucous neurons to the myenteric plexus in the guinea pig small intestine

The distribution of submucous neurons that project to the myenteric plexus of the guinea pig small intestine was established by retrograde transport of the carbocyanine dye 1,1′-didodecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (DiI) from myenteric ganglia in organ culture in combination with immunohistochemistry. Following the application of DiI to the serosal surface of a single myenteric ganglion, from 2 to 15 DiI-labelled nerve cell bodies were labelled in the submucous plexus up to 7.9 mm circumferentially, 4.5 mm orally, and 3.4 mm aborally to the DiI application site. No cells were labelled in preparations in which connections between myenteric and submucous plexuses had been severed prior to DiI application. Cells that were immunoreactive for vasoactive intestinal polypeptide (VIP) or for substance P (SP) accounted for about 75% and 11% of DiI-labelled cells, respectively. Neither neuropeptide Y- nor calretinin-immunoreactive submucous neurons were labelled by DiI, indicating that these classes of neurons do not project to the myenteric plexus. Retrograde tracing from the myenteric plexus with Neurobiotin revealed that labelled VIP-immunoreactive neurons had several short, filamentous processes and a single long axon that could be followed through the circular muscle to myenteric ganglia without branches to the mucosa. The previously described projection of submucous, SP-immunoreactive putative sensory neurons to the myenteric plexus was confirmed. However, this study has identified a considerably larger population of presumed interneurons that are immunoreactive for VIP that likely transmit information from the submucous plexus to the myenteric plexus and presumably coordinate activity between the two ganglionated plexuses. J. Comp. Neurol. 399:255–265, 1998. © 1998 Wiley-Liss, Inc.     

Synaptic transmission from the submucosal plexus to the myenteric plexus in Guinea‐pig ileum

Abstract Stimulation of the myenteric plexus results in activation of submucosal neurons and dilation of arterioles, one way that motility and secretion can be coupled together. The present study aimed to examine the converse, whether myenteric neurons receive synaptic input from the submucosal plexus (SMP). Intracellular recordings were made from guinea‐pig ileal myenteric neurons while the SMP was electrically stimulated. Of the 29 neurons studied (13 S and 16 AH neurons), stimulation of the SMP evoked a synaptic potential in only seven cells, or 24% of neurons. When the SMP was situated oral to the myenteric plexus, 4 of 13 (31%) myenteric neurons had synaptic input. When it was situated circumferential, 2 of 8 (25%) had input, and when the SMP was situated anal 1 of 8 (13%) had input. Overall, 5 of the 13 (38%) S neurons responded with fast excitatory post‐synaptic potentials (EPSPs), one of which also showed a slow EPSP, while 2 of the 16 (13%) AH neurons responded with a slow EPSP. This study indicates that the synaptic input from the SMP to myenteric neurons is relatively sparse. Whether this input is less important than the myenteric to submucosal input or simply represents a more selective form of control is unknown.     

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon

Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to control of blood flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic, and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter, and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic, and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6), and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide. They had distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies. J. Comp. Neurol. 523:742–756, 2015. © 2014 Wiley Periodicals, Inc.     

Encephalomyeloneuropathy with ganglionitis of the myenteric plexuses in the absence of cancer

A 55-year-old woman presented with rapidly progressive brainstem dysfunction which led to death within a month. She also had constipation for three weeks, and barium enema showed ileus. Subacute encephalomyelitis predominantly involving the medulla and pons correlated with the patient's initial symptoms. In addition, ganglionitis of the myenteric plexuses explained the constipation and ileus. Ganglioradiculoneuropathy was another finding. The presence of abundant neuronophagia in the brainstem, dorsal root ganglia, and myenteric plexuses-raised the speculation that a putative virus, toxic agent, or immune reaction possessed special affinity for neurons and ganglion cells. The neuropathological findings were similar to paraneoplastic changes, but no neoplasm was found.     

Neurochemically distinct classes of myenteric neurons express the mu-opioid receptor in the guinea pig ileum

The mu-opioid receptor (muOR), which mediates many of the opioid effects in the nervous system, is expressed by enteric neurons. The aims of this study were to determine whether 1) different classes of myenteric neurons in the guinea pig ileum contain muOR immunoreactivity by using double- and triple-labeling immunofluorescence and confocal microscopy, 2) muOR immunoreactivity is localized to enteric neurons immunoreactive for the endogenous opioid enkephalin, and 3) muOR immunoreactivity is localized to interstitial cells of Cajal visualized by c-kit. In the myenteric plexus, 50% of muOR-immunoreactive neurons contained choline acetyltransferase (ChAT) immunoreactivity, whereas about 43% of ChAT-immunoreactive neurons were muOR immunoreactive. Approximately 46% of muOR myenteric neurons were immunoreactive for vasoactive intestinal polypeptide (VIP), and about 31% were immunoreactive for nitric oxide synthase (NOS). MuOR immunoreactivity was found in about 68% of VIP-containing neurons and 60% of NOS-immunoreactive neurons. Triple labeling showed that about 32% of muOR neurons contained VIP and ChAT immunoreactivities. The endogenous opioid enkephalin (ENK) was observed in about 30% of muOR neurons; conversely, 48% of ENK neurons contained muOR immunoreactivity. MuOR was not detected in neurons containing calbindin, nor in interstitial cells of Cajal. MuOR-immunoreactive fibers formed a dense network around interstitial cells of Cajal in the deep muscular plexus. This study demonstrates that muOR is expressed by neurochemically distinct classes of myenteric neurons that are likely to differ functionally, is colocalized with the endogenous opioid ENK, and is not expressed by interstitial cells of Cajal.     

Electrochemical monitoring of nitric oxide released by myenteric neurons of the guinea pig ileum

Abstract Nitric oxide (NO) released by myenteric neurons in isolated segments of guinea pig ileum was monitored in vitro using continuous amperometry. NO was detected as an oxidation current recorded with a boron‐doped diamond microelectrode held at 1 V vs a Ag|AgCl reference electrode. This potential was sufficient to oxidize NO. Longitudinal muscle‐myenteric plexus (LMMP) and circular muscle strip preparations were used. In the LMMP preparation, NO release was evoked by superfusion of 1 μmol L^?1 nicotine, which activates nicotinic acetylcholine receptors expressed by myenteric neurons and myenteric nerve endings. The oxidation current was ascribed to NO based on the following observations: (i) no response was detected at less positive potentials (0.75 V) at which only catecholamines and biogenic amines are oxidized, (ii) the current was abolished in the presence of the nitric oxide synthase antagonist, N‐nitro‐l ‐arginine (l ‐NNA) and (iii) oxidation currents were attenuated by addition of the NO scavenger, myoglobin, to the superfusing solution. In the LMMP preparation, stimulated release produced a maximum current that corresponded nominally to 46 nmol L^?1 of NO. The oxidation currents decreased to 10 and 2 nmol L^?1, respectively, when the tissue was perfused with tetrodotoxin and l ‐NNA. Oxidation currents recorded from circular muscle strips (stimulated using nicotine) were threefold larger than those recorded from the LMMP. This study shows that NO release can be detected from various in vitro preparations of the guinea pig ileum using real‐time electroanalytical techniques.     

Morphology, electrophysiology, and calbindin immunoreactivity of myenteric neurons in the guinea pig distal colon

The morphological and physiological characteristics of myenteric neurons in the guinea pig distal colon were determined using Lucifer yellow- or N-(2-aminoethyl) biotinamide-containing microelectrodes and intracellular recording and staining methods. The neurons in this study (n = 204) were classified on the basis of the shapes of their cell bodies and short processes or dendrites and the number of long processes or axons as Dogiel type I (n = 75 neurons; 36.8%), filamentous (n = 31 neurons; 15.2%), Dogiel type II (n = 38 neurons; 18.6%), and unclassified (n = 60 neurons; 29.4%). All Dogiel type II neurons had action potentials followed by an after-spike hyperpolarization (AH), and most of them (84%) had large, smooth somata and filamentous, short processes in addition to multiple, long processes or axons. Most of Dogiel type I, filamentous, and unclassified neurons (98%) had a single, long process, but four Dogiel type I neurons and one unclassified neuron had two long processes terminating as varicosities within other ganglia or on the surface of longitudinal muscle. The projections of monoaxonal neurons were distributed equally between oral and aboral directions, and most of them received fast excitatory postsynaptic potentials (EPSPs). All of the Dogiel type II neurons and seven Dogiel type I neurons were positive for calbindin immunoreactivity, but three filamentous neurons received fEPSPs, had spikes followed by AH, and were negative for calbindin. The presence of calbindin-immunoreactive(-IR) neurons was quite variable among the ganglia. These results confirm that neither the presence of calbindin immunoreactivity nor the absence of fEPSPs can be used as a predictor of cellular morphology or electrophysiological properties of myenteric neurons in the distal colon.     

Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation

We studied the characteristics of electrical coupling between Bergmann glial cells in mouse cerebellar slices using Lucifer Yellow injection, patch-clamping cell pairs, and ultrastructural inspection. While early postnatal cells (days 5–7) were not coupled, coupling was abundant at postnatal days 20–24. Coupled cells were arranged perpendicular to the parallel fibers in a parasagittal section, forming a string, rather than a cluster of cells. Electron microscopy revealed that gap junctions were abundant in the distal parts of the processes. Gap junctions between cell bodies and processes were very rare, and no gap junctions were found between cell bodies of adjacent Bergmann glial cells. The junctional conductance was voltage and time independent and could be markedly reduced by halothane. Alkalization of cells (by applying NH₄⁺ increased the junctional conductance to 150%, while acidification of the cell interior (by removing NH₄⁺) led to a decrease to 70%. Activation of AMPA receptors induced a blockade of the junctional conductance to 30% of the control. This link is most likely mediated by the influx of Ca²⁺ via the receptor since this effect was not observed in Ca²⁺-free medium, suggesting that Ca²⁺ entry via the kainate receptor pore led to the closure of gap junctions. These studies indicate that electrical coupling between Bergmann glial cells is not only developmentally regulated but also controlled by physiological stimuli. © 1996 Wiley-Liss, Inc.     

Light stimulation evokes two different calcium responses in Müller glial cells of the guinea pig retina

Intracellular calcium responses are a characteristic of glial activation upon neuronal activity. In acutely isolated preparations of the guinea pig retina, Müller glial cells displayed cytosolic calcium rises in response to repetitive light stimulation. The calcium rises consisted of two components, a slowly developing immediate response that occurred simultaneously over the whole length of all Müller cell fibers and a delayed fast response that originated in the ganglion cell layer and spread as a wave through the bodies of some Müller cells toward the outer processes in the photoreceptor layer. The slow calcium response was evoked by photoreceptor-to-glia signaling, resulting in a glutamate transporter- and zinc-mediated alteration in the membrane potential and an influx of calcium from the extracellular space. The fast calcium response was evoked by a release of calcium from intracellular stores, probably after activation of purinergic receptors. The data suggest that light stimulation of the retina causes glial activation by alterations in both the membrane potential and receptor-mediated mechanisms. The former may be implicated in glial support of the neuronal signal transfer from photoreceptors to ganglion cells (glial forward signaling), whereas the latter may constitute a glial feedback signaling from ganglion cells to photoreceptors.     

S100 on isolated neurons and glial cells from rat, rabbit and guinea pig during early postnatal development.

The brain specific S100 protein has been demonstrated by immunofluorescence microscopy on neuronal cell membranes during postnatal development of rat, rabbit and guinea pig. S100 has been found in glial cell aggregates of rats 4-5 days old, of rabbits 2-3 days old, and of newborn guinea pig. The neuronal plasma membrane-bound portion of the protein appears later during development. In rats 10 to 12 days of age S100 could be found on one part of the cell membrane of Deiters' neurons and of Purkinje cells. In rats 12 to 15 days old cells from the cerebral cortex contained a heterogeneously distributed cell membrane-bound portion of S100. The protein on neuronal cell membranes could be seen somewhat earlier in rabbits as compared to rats. Newborn guinea pigs showed a heterogeneous distribution of the protein similar to that seen in adult animals. In rat and rabbit adults distribution and amounts of S100 were reached at days 25-30 and at days 20-24, respectively. The results obtained here are in agreement with biochemical results. Our findings suggest that the membrane-bound part of the S100 protein with its heterogeneous and polar distribution on the nerve cell plasma membranes (demonstratable in parallel with physical and behavioral maturation of the animals) is a sign of a protein differentiation of the neuron.