Localization of calmodulin in rat cerebellum by immunoelectron microscopy.

Calmodulin, a multifunctional Ca(++)-binding protein, is present in all eucaryotic cells. We have investigated the distribution of this protein in the rat cerebellum by immunoelectron microscopy using a Fab-peroxidase conjugate technique. In Purkinje and granular cell bodies, calmodulin reaction product was found localized both on free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No calmoduline was observed in the cisternae of RER or the Golgi apparactus. Calmodulin did not appear to be concentrated in the soluble fraction of the cell under the conditions used. Rather, peroxidase reaction product could be seen associated with membranes of the Golgi apparatus the smooth endoplasmic reticulum (SER), and the plasma membrane of both cell bodies and neuronal processes. In the neuronal dendrites, calmodulin appeared to be concentrated on membranes of the SER, small vesicles, and mitochondria. Also, granular calmodulin was observed in the amorphous material. In the synaptic junction, a large amount of calmodulin was seen attached to the inner surface of the postsynaptic membrane, whereas very little was observed in the presynaptic membrane or vesicles. These observations suggest that calmodulin is synthesized on ribosomes and discharged into the cytosol, and that it then becomes associated with a variety of intracellular membranes. Calmodulin also seems to be transported via neuronal processes to the postsynaptic membrane. Calmodulin localization at the postsynaptic membrane suggests that this protein may mediate calcium effects at the synaptic junction and, thus, may play a role in the regulation of neurotransmission.

Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells.

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.

Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy.

Microtubule-associated protein 1A (MAP1A) and microtubule-associated protein 2 (MAP2) were shown to be colocalized on the same microtubules (MTs) within neuronal cytoskeletons by double-label immunoelectron microscopy. To investigate the electron microscopic disposition of MAP1A and MAP2 and their relationship to MTs in vivo, and to determine whether there are different subsets of MTs which specifically bind either MAP1 or MAP2, we employed a double-label immunogold procedure on rat cerebella using mouse monoclonal antibody against rat brain MAP1A and affinity-purified rabbit polyclonal antibody against rat brain MAP2. MAP1A and MAP2 were identified with secondary antibodies coupled to 10- and 5-nm gold particles, respectively. In Purkinje cell dendrites, both 10- and 5-nm gold particles were observed to be studded on the fuzzy structures attached to the same MTs. Many such structures connected MTs to each other. There was no particular MT which bound either MAP1A or MAP2 alone. Furthermore, there seemed to be no specific regions on MTs where either MAP1A or MAP2 was specifically attached. Hence, we conclude that MAP1A and MAP2 are colocalized on MTs in dendrites and assume that MAP1A and MAP2 have some interrelationship in vivo and that their interactions are responsible for forming the network of cross-bridges between MTs and MTs in neuronal cytoskeletons.

Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain.

Antisera to calmodulin, a Ca2%-dependent modulator protein, and a heat-labile calmodulin-binding protein have been used to localize these proteins in mouse caudate-putamen. The two proteins appear to be located at identical sites in this brain area. At the light microscopic level, calmodulin and calmodulin-binding protein are found within the cytoplasm and processes of large cells. At the electron microscopic level the proteins are associated with neuronal elements only, primarily at postsynaptic sites within neuronal somata and dendrites. Within the dendrites the immunocytochemical label is associated predominantly with the postsynaptic density and dendritic microtubules. These results are in accord with recent biochemical and immunihistochemical studies of calmodulin in brain and in dividing cells. Thus, calmodulin and the heat-labile calmodulin-binding protein may play a role in the nervous system at the site of neurotransmitter action and at the level of microtubular function.

Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling.

Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum.

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.

Immunoelectron microscopic localization of the neural cell adhesion molecules L1 and N-CAM during postnatal development of the mouse cerebellum.

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.

Identification and localization of ryanodine binding proteins in the avian central nervous system.

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of approximately 2000 kd and are composed of two high molecular weight (approximately 500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.

Alterations in Langerhans cells and Thy-1+ dendritic epidermal cells in murine epidermis during the evolution of ultraviolet radiation-induced skin cancers.

To understand the role of cutaneous immune cells in host resistance to the induction and growth of skin cancer, we investigated the number and morphology of murine dendritic epidermal cells (dEC) during the evolution of ultraviolet (UVA) UV-induced skin cancers. Female C3H/HeN mice were treated topically with 8-methoxypsoralen followed by ultraviolet A (UVA) radiation 3 times/week or irradiated with UVB radiation 3 times/week. In both psoralen plus UVA- and UVB-treated mice, ATPase+ and Ia+ Langerhans cells almost completely disappeared from the treated skin during the early latency period of tumor development (4 weeks) but reappeared in the epidermis late in the latency period (between 15 and 22 weeks). The ATPase+ cells that reappeared in the epidermis had a rounder, less dendritic morphology than normal Langerhans cells. Thy-1+ dEC were totally depleted from the epidermis in both treatment groups at the end of first week of treatment and were nearly absent from the skin during the entire latency period. After tumors appeared (29 weeks), Thy-1+ dEC were still absent or detected only in small numbers in skin surrounding the tumors. ATPase+ and Ia+ cells present in skin around the tumors constituted 60 to 80% of the number in nonirradiated skin. Mice that received UVA radiation alone developed no tumors. ATPase+ and Ia+ Langerhans cells and Thy-1+ dEC were detected in UVA-treated epidermis after 22 weeks and 43 weeks, although the numbers were lower than those in unirradiated mice. Most psoralen plus UVA-induced tumors (81%) were squamous cell carcinomas, whereas only 24% of UVB-induced tumors were of this histological type. Our results demonstrate that UV-induced skin cancers developed in the presence of ATPase+ and Ia+ cells in the epidermis and in the absence of Thy-1+ dEC.

An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture.

We have studied the development of intermediate filament proteins in the neurons found in hippocampal cell cultures using single and double label immunofluorescence with both monoclonal and polyclonal antibodies. Neurons in these cultures are known to differentiate in a manner similar to their counterparts in situ: in particular they develop axonal and dendritic processes which differ from each other in form, in ultrastructure, and in synaptic polarity. During the first days in culture, developing neurons could not be stained with antibodies against any of the neurofilament proteins, although many cells reacted with anti-vimentin. Later in the first week, antibody staining revealed clearly filamentous staining for the L (68 000 daltons) and the M (145 000 daltons) neurofilament subunits, though M reactivity was much stronger at this earlier stage of development. Some neurofilament positive profiles in many cells could also be stained with vimentin, though the vimentin immunoreactivity became progressively less pronounced during further development, and disappeared after about two weeks in culture. Also at about two weeks in vitro we noted the first appearance of neurofilament H protein (200 000 daltons) immunoreactivity, which was localized to a subset of long neurites which could be identified on morphological grounds as axons. These processes lacked staining for microtubule associated protein 2 (MAP2), a dendritic marker. They tended to be close to islands of glial cells, suggesting that H induction may require complex neuron-glial interactions. These results are consistent with the suggestion that H protein immunoreactivity is a marker for axonal outgrowth. In addition to obvious filamentous staining, we were able to localize neurofilament antigens to an interesting class of small ring-like structures, found increasingly frequently as the cultures aged. We also present evidence that tyrosinated alpha-tubulin is present both within dendrites and axons of neurons in these cultures.

Effect of ultraviolet B radiation on S-100 protein antigen in epidermal Langerhans cells.

Ultraviolet B (UVB) radiation has been shown to induce significant alterations in both function and surface antigen expression of epidermal Langerhans cells (ELC). In this study we investigated the effect of UVB radiation on ELC marker S-100 protein antigen (S-100 Ag) which is present in the nucleus and cytoplasm of human ELC. A total of 34 sites on 31 volunteers were exposed to 3 MED (minimal erythema dose) of UVB and biopsied at various times up to 7 days after irradiation. Skin from 9 noninjured and 7 slice-wounded subjects served as controls. The avidin-biotin-peroxidase staining technique was used to identify S-100 Ag in sections of formalin-fixed, paraffin-embedded tissue, and the numbers of stained suprabasal dendritic cells were then counted over a 200 basal cell length of interfollicular epidermis. Noninjured skin had 3.56 +/- 3.01 cells, whereas slice-wounded skin had elevated numbers (greater than 10.0 cells) at 1, 24, and 48 h after injury. Following UVB irradiation, a significant (p less than 0.001) increase in antigen-positive cells (14 +/- 3.46) was found at 1 h; this number declined to just below normal at 12 h, but by 48 h returned to and remained at preinjury levels. In contrast to previous observations of the depletion of ELC surface markers by UVB radiation, we demonstrate here that the numbers of S-100 Ag-positive ELC actually increase following comparable doses of radiation. Since this increase occurs so rapidly following both UVB irradiation and slice injury, S-100 Ag may be synthesized or unmasked within the ELC as a response to wounding of the epidermis.

The intercalated cells of the amygdala.

The intercalated cell groups, or massa intercalata, of the amygdala have been studied in rodent brains with Golgi methods. They also have been examined in gallocyanin-chromalum-, AChE-, and Timm-stained rat brains. The Golgi data indicate that the intercalated cells are not confined to a series of isolated cell clumps but form a neuronal net that covers the rostral half of the lateral-basolateral nuclear complex, stretches across a major portion of rostral amygdala, and continues rostrally beneath the anterior commissure. There are two general types of intercalated neuron--medium and large neurons. The medium intercalated neurons are more common. They have round to elongate somata, 9-18 microns in diameter, and round to bipolar dendritic trees, depending on their location. Most of the dendrites are spine-bearing, as are 20% of the somata. Their axons often have locally ramifying collaterals. The parent axons apparently terminate in either the lateral-basolateral or central nuclei and some of them appear to enter the external capsule. There is a unique medium intercalated neuron that has nearly spine-free, varicose dendrites and an axon that is typical of short axon (Golgi II) cells. There are two varieties of large intercalated neuron-spiny and aspiny. Most of them are aspiny, although they usually have a few spines scattered along their dendrites. Both varieties have elongate, sometimes round, somata that can be as much as 60 microns long. Their dendrites are long, thick, and have few branch points. Only the initial part of the large aspiny cell axon has been impregnated. The large spiny cell axons have several local collaterals; the destination of the parent axons is unknown. The intercalated cells occur along fiber bundles, which are probably afferent to them. The axons that travel among the intercalated cells give off short collaterals and boutons en passant. The sources of these fibers are not known. From the published experimental data, it is likely that they originate in the piriform and entorhinal cortices, the lateral preoptic area, lateral hypothalamus, and ventral pallidum. Axon collaterals of basolateral nucleus pyramidal cells appear to terminate among the intercalated cells. It is suggested that the intercalated cells serve as sites for integration of the output of these various areas and, in turn, communicate it to the lateral-basolateral and central amygdaloid nuclei. The intercalated cells closely resemble neurons in the corpus striatum. Thus the question is raised and discussed of whether the intercalated cells are a ventral extension of the corpus striatum.

A Golgi analysis of the gustatory zone of the nucleus of the solitary tract in the adult hamster.

The somal shapes, dendritic features, and orientations of the neurons within the gustatory zone of the nucleus of the solitary tract were studied with the rapid Golgi method in the adult hamster. These Golgi studies complement previous quantitative morphometric analyses of the distributions of large and small neurons within the gustatory zone. Class 1 neurons are usually fusiform and possess long, relatively unbranched dendrites that often extend beyond the cytoarchitectonic boundaries of the gustatory zone. Class II neurons are multipolar and possess more dendrites that are significantly shorter than those of class I neurons. Both classes of neurons are spine poor. Computer-generated three-dimensional rotational analyses demonstrate that the dendritic arborizations of neurons of the gustatory zone are oriented preferentially in the horizontal plane. Dendrites extend in parallel or perpendicular to the solitary tract, the source of peripheral gustatory inputs, and appear to be positioned spatially to maximize synaptic interactions with these peripheral fibers. These Golgi studies also suggest that individual gustatory neurons may be influenced by incoming gustatory fibers that innervate separate populations of taste buds, a finding that is not predictable from the topographical organization of the gustatory zone.

Localization of tyrosine-hydroxylase-like-immunoreactive amacrine cells in the larval tiger salamander retina.

Immunocytochemistry was used to localize the populations of tyrosine-hydroxylase-like (TH)-immunoreactive cells in the tiger salamander retina. Ninety percent of these cells possessed somas that were situated in the innermost cell row of the inner nuclear layer and were classified as amacrine cells. Ten percent of TH-immunoreactive somas were located in the ganglion cell layer and were tentatively designated as those of displaced amacrine cells. The processes of TH-immunoreactive cells ramified most heavily in sublayer 1 of the inner plexiform layer, while a relatively small number of TH-labelled processes distributed in sublayers 3 and 5. Less than 1% of TH-immunoreactive cells in the amacrine cell layer exhibited a short process of somal origin that extended distally toward the outer plexiform layer. However, these processes did not cross the whole of the inner nuclear layer, and no immunolabelling was observed in the outer plexiform layer. An examination of retinal whole-mounts revealed that TH-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and periphery of the retina. The density of TH-immunolabelled amacrine cells was calculated to be 49 +/- 13 (mean +/- standard error) cells per mm2. The vast majority of TH-immunoreactive amacrine and displaced amacrine cells exhibited a stellate appearance and gave rise to three or more primary dendrites. A few TH-amacrine and displaced amacrine cells possessed two primary dendrites that emerged from opposite sides of their somas. The processes of TH-immunoreactive cells were generally poorly branched and varicose with terminal branches sometimes appearing thin and beaded. Because some TH-immunolabelled processes were very long, there was considerable overlap between the dendritic fields of neighboring TH-cells. Lastly, individual TH-immunoreactive amacrine and displaced amacrine cells were often observed in whole-mounts to provide processes that ramified at more than one level of the inner plexiform layer.

Morphologies of rabbit retinal ganglion cells with concentric receptive fields.

Rabbit retinal ganglion cells with concentric receptive fields were intracellularly recorded and stained in the isolated superfused eyecup preparation to relate specific physiological response properties to dendritic morphology. Concentric ganglion cells, as traditionally defined, were those that had On or Off centers with antagonistic surrounds but lacked complex response properties such as direction or orientation selectivity. Concentric cells were classified into different groups by extracellular recordings of their On- or Off-center response sign, excitatory receptive field center size, linearity of spatial summation, and brisk vs. sluggish and transient vs. sustained responses to step changes in light intensity. The cells were then impaled, confirmed in identity during intracellular recording, and iontophoretically injected with horseradish peroxidase for histological analysis. Twenty-three concentric ganglion cells were recovered and morphometrically analyzed. Their physiological response properties were found to be related to a number of underlying two- and three-dimensional attributes of the cell's dendritic branching patterns. The dendrites of all 20 brisk concentric cells and two of the three sluggish cells were found to ramify narrowly in either the proximal or distal half of the inner plexiform layer, corresponding to whether they are On center or Off center, respectively. One of the sluggish concentric cells was found to have a more complex, partially bistratified ramification. Physiologically identified brisk-sustained-linear, brisk-transient-nonlinear, brisk-transient-linear, and at least two classes of sluggish concentric ganglion cells were stained. Each of these physiological classes appears to exhibit a distinct and identifiable dendritic branching pattern.

Morphologies of rabbit retinal ganglion cells with complex receptive fields.

Ganglion cells that had complex receptive field properties, namely, On-Off and On direction-selective cells, orientation-selective cells, local edge detectors, and uniformity detectors (suppressed by contrast cells) were recorded in an isolated superfused rabbit eyecup preparation. Cells were first classified by their characteristic extracellular responses to manually controlled stimuli similar to those which have been used in previous in vivo studies. Ganglion cells were then impaled, confirmed in identity by intracellular recording, and iontophoretically injected with horseradish peroxidase for staining. Twenty-two ganglion cells, which included members of all the major classes mentioned above, were recovered from the visual streak or near periphery. All recovered cells were drawn in camera lucida from flat-mounted retinas and entered into a computer as two-dimensional stick figures; nearly all were three-dimensionally reconstructed to determine the level and manner of dendritic ramification in the inner plexiform layer (IPL). The location of ganglion cell dendrites in sublaminar regions of the IPL was found to be consistent with the hypothesis of a division of the IPL into excitatory On (proximal) and Off (distal) sublaminae, with some qualifications for particular classes. Each of the complex receptive field ganglion cell classes exhibited a distinctive three-dimensional dendritic arborization pattern uniquely associated with that physiological class.

Morphometric analysis of serotoninergic bipolar cells in the retina and its implications for retinal image processing.

The entire population of retinal serotoninergic bipolar cells in the turtle Pseudemys scripta elegans was labeled by immunocytochemical methods. This allowed a systematic analysis to be made of the morphological variabilities among a functionally homogeneous neuronal population. The analyzed morphological characteristics included: size of the Landolt club, size of the soma, lateral extension of the ramification within the outer plexiform layer, course of the axon across the inner nuclear layer, pattern of the axonal ramification within the inner plexiform layer, lateral extension of these ramifications, and density of the cells. Whereas characteristics 1-4 and 7 show a morphological variability strictly related to the location of the bipolar cell with respect to the visual streak, a fovealike structure, characteristics 5 and 6 show no such correlation. The size of the soma increases by a factor of 4 from the visual streak toward the periphery. The area covered by the ramification in the OPL increases from 330 micron 2 at the visual streak to 50,000 microns 2 at the dorsal and ventral edges of the retina. The coverage factor remains the same throughout the retina, as well as the area covered by the ramifications in the IPL, which is about 2,000 microns 2. At the visual streak and at 110 degrees ventral and 68 degrees dorsal of the visual streak, the bipolar axons cross the INL perpendicularly. In between the axons take an oblique course leading to an axon-induced shift between input and output of up to 250 microns.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal development of lamina III/IV nonpyramidal neurons in rabbit auditory cortex: quantitative and spatial analyses of Golgi-impregnated material.

We have studied the postnatal development of lamina III/IV spine-free nonpyramidal neurons in the auditory cortex of the New Zealand white rabbit. The morphology and dendritic branching pattern of single cells impregnated with a Golgi-Cox variant were analyzed with the aid of camera lucida drawings and three-dimensional reconstructions obtained with a computer microscope. Sample sizes of 20 neurons were obtained at birth (day 0), postnatal day (PD) 3, 6, 9, 12, 15, 21, and 30 days of age. Normative data were also available from PD-60 and young adult rabbits studied previously. At birth, lamina II-IV have not yet emerged from the cortical plate; immature nonpyramidal neurons at the bottom of the cortical plate (presumptive layer IV) are characterized by short, vertically oriented dendrites. Growth-cone-like structures are present along the shafts and at the tips of the dendrites. At birth, soma area and total dendritic length are, respectively, 34 and 10% of adult values. The cortical plate acquires a trilaminar appearance at PD-3. The six-layered cortex is present by PD-6. During the first postnatal week dendritic length increases fourfold and is accompanied by a significant increase in both terminal and preterminal dendritic growth cones. At the onset of hearing at PD-6, there is a significant proliferation of dendrites and branches to 144 and 200% of adult levels, respectively. These supernumerary dendrites are rapidly lost during the second postnatal week, at which time the somata and dendrites become covered with spines. The loss of higher-order dendrites occurs more gradually; the number of dendritic branches is still 116% of adult values at PD-30. Spine density peaks between days PD-12 and PD-15, and then gradually diminishes until the cells are sparsely spined or spine free by PD-30. Total dendritic length increases in a linear fashion up to PD-15, at which time it is 80% of adult values. An analysis of terminal and intermediate branches demonstrated that the increase in total dendritic length after PD-6 is due entirely to the growth of terminal dendrites. Total dendritic length attains adult levels by PD-30. Spatial analyses revealed that a vertical orientation of dendrites is present at birth. Associated with the onset of hearing at PD-6, there is an explosive elaboration of dendrites toward the pial surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal development of cat hind limb motoneurons. III: Changes in size of motoneurons supplying the triceps surae muscle.

The postnatal changes of neuronal dimensions were studied in cat triceps surae motoneurons intracellularly labeled with horseradish peroxidase. Systematic correlations were observed in the analysis of single dendrites at each studied stage, from birth to 44-46 days post natum (d.p.n.) age, between size parameters intrinsic to the dendrites as the diameter of a 1st-order dendrite, the combined dendritic length, the dendritic membrane area, and the degree of branching. Some variability among samples was evident in each studied age group. The correlations were, however, sufficiently close to permit indirect estimations of both combined dendritic length and dendritic membrane area for larger samples of neurons from data on dendritic stem caliber. The total postnatal increase in dendritic membrane area was, on the average, 400%, i.e., from close to 100 X 10(3) microns2 to about 500 X 10(3) microns2. The corresponding increase in soma area amounted to 100%. Analysis revealed that there was a time lag between the increase in somatic and dendritic size. Thus, adult somatic dimensions were attained at age 44-46 d.p.n.; however, at this stage, the mean total dendritic membrane area was only about half of the adult value. The postnatal increase in size appeared to vary among neurons, yielding a wider neuronal size spectrum in the adult cat than that observed in kittens. The measured increase in size corresponded to a calculated average addition of dendritic membrane area of 3700 microns2/day from birth to 22-24 d.p.n. and from that stage to 44-46 d.p.n. of 2700 microns2 per day. Likewise, the increase in combined dendritic length could initially be as large as 1 mm/day down to 0.4 mm/day between 22-24 and 44-46 d.p.n., with a mean growth during the first 44-46 d.p.n. of 0.5 to 0.6 mm/day. The ratios of daughters to parent branch diameters (sigmadd1.5: dp1.5) and the dendritic trunk parameter (sigma d1.5) recorded along the proximodistal dendritic path distance revealed transient changes that might impact on the electrotonic properties of the dendrites during postnatal development. Computations from the measured changes in dendritic branch lengths and calibers indicated that if membrane and internal resistivity remain unaltered during postnatal development, the dendritic domain is electrotonically more compact in the newborn kitten than in the adult cat.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for the inhibitory neurotransmitter gamma-aminobutyric acid in aspiny and sparsely spiny nonpyramidal neurons of the turtle dorsal cortex.

In order to learn more about the anatomical substrate for gamma-aminobutyric acid (GABA)-mediated inhibition in cortical structures, the intrinsic neuronal organization of turtle dorsal cortex was studied by using Golgi impregnation, immunohistochemical localization of GABA and its synthetic enzyme glutamic acid decarboxylase (GAD), and histochemical localization of the presynaptic GABA-degrading enzyme GABA-transaminase (GABA-T). GABAergic markers are found in neurons identical in morphology and distribution to Golgi-impregnated aspiny and sparsely spiny nonpyramidal neurons with locally arborizing axons and appear to label most if not all of the nonpyramidal neurons. In addition, the GABAergic markers are found in punctate structures in a distribution characteristic of presumed inhibitory terminals. The spine-laden pyramidal neurons, the principal projecting cell type in the dorsal cortex, are devoid of labelling for GABAergic markers but are surrounded by presumed GABAergic terminals. The data complement previous physiological and ultrastructural studies that implicate aspiny and sparsely spiny nonpyramidal neurons as mediators of intrinsic inhibition of pyramidal neurons in turtle cortex. The results also suggest similarities in the functional organization of intrinsic inhibitory elements in turtle and mammalian cortex.