Sound generation due to the interaction of turbulence with surfaces embedded in transversely sheared flow.

This paper reviews the application of Rapid Distortion Theory (RDT) on transversely shear mean flows to the prediction of sound generated from solid surfaces imbedded in turbulent shear flows. This phenomenon is relevant to the so-called installation noise problem which has received considerable attention in recent years. A few representative results from applications that have appeared in the literature are also presented. This article is part of the theme issue 'Frontiers of aeroacoustics research: theory, computation and experiment'.

Nonlinear wakes behind a row of elongated roughness elements.

This paper is concerned with the high Reynolds number flow over a spanwise-periodic array of roughness elements with interelement spacing of the order of the local boundary-layer thickness. While earlier work by Goldstein et al. (J. Fluid Mech., vol. 644, 2010, pp. 123-163) and Goldstein et al. (J. Fluid Mech., vol. 668, 2011, pp. 236-266) was mainly concerned with smaller roughness heights that produced relatively weak distortions of the downstream flow, the focus here is on extending the analysis to larger roughness heights and streamwise elongated planform shapes that together produce a qualitatively different, nonlinear behaviour of the downstream wakes. The roughness scale flow now has a novel triple-deck structure that is somewhat different from related studies that have previously appeared in the literature. The resulting flow is formally nonlinear in the intermediate wake region, where the streamwise distance is large compared to the roughness dimensions but small compared to the downstream distance from the leading edge, as well as in the far wake region where the streamwise length scale is of the order of the downstream distance from the leading edge. In contrast, the flow perturbations in both of these wake regions were strictly linear in the earlier work by Goldstein et al. (2010, 2011). This is an important difference because the nonlinear wake flow in the present case provides an appropriate basic state for studying the secondary instability and eventual breakdown into turbulence.

Effect of free-stream turbulence on boundary layer transition.

This paper is concerned with the transition to turbulence in flat plate boundary layers due to moderately high levels of free-stream turbulence. The turbulence is assumed to be generated by an (idealized) grid and matched asymptotic expansions are used to analyse the resulting flow over a finite thickness flat plate located in the downstream region. The characteristic Reynolds number Rλ based on the mesh size λ and free-stream velocity is assumed to be large, and the turbulence intensity ε is assumed to be small. The asymptotic flow structure is discussed for the generic case where the turbulence Reynolds number εRλ and the plate thickness and are held fixed (at O(1) and O(λ), respectively) in the limit as [Formula: see text] and ε→0. But various limiting cases are considered in order to explain the relevant transition mechanisms. It is argued that there are two types of streak-like structures that can play a role in the transition process: (i) those that appear in the downstream region and are generated by streamwise vorticity in upstream flow and (ii) those that are concentrated near the leading edge and are generated by plate normal vorticity in upstream flow. The former are relatively unaffected by leading edge geometry and are usually referred to as Klebanoff modes while the latter are strongly affected by leading edge geometry and are more streamwise vortex-like in appearance.

Differential expression of the alpha and beta subunits of the large-conductance calcium-activated potassium channel: implication for channel diversity.

In addition to the large alpha subunits that conduct selective ion currents, many native voltage-gated ion channels contain associated proteins which modulate the channel activity. Recently, a beta subunit of the large-conductance calcium-activated K+ (BK) channel has been cloned and functionally characterized. In this report, we studied the tissue distribution of the alpha and beta subunits of rat BK channels by nuclease protection analyses and in situ hybridization. BK alpha mRNA is widely distributed but is especially enriched in the brain. In the adult brain, BK alpha expression is robust and widespread throughout all areas of the neo-, olfactory and hippocampal cortices, habenula and cerebellum. Other prominent sites of BK alpha expression include thalamus and amygdala. In marked contrast to the expression pattern of BK alpha mRNA, the expression of BK beta mRNA is relatively low and preferentially in the periphery. In rat brains, BK beta mRNA occurs only in a few discrete populations of neurons that also express BK alpha messages. These results indicate that the major type of BK channels in the brain, unlike the alpha beta channel type in aortic and tracheal smooth muscle, is devoid of the beta subunit. These observations provide a structural basis for the BK channel diversity observed in a variety of tissues.

Cloning, pharmacological characterization and distribution of a novel galanin receptor.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G-protein-coupled receptors. Through expression cloning, human and rat GALR1 receptor cDNA clones have previously been isolated and characterized. In this study, we have used homology screening to isolate a rat brain cDNA clone encoding a second galanin receptor subtype, the GALR2 receptor. The isolated cDNA encodes a 372-amino-acid G-protein-coupled receptor that shares 38% overall amino-acid identity with the rat GALR1 receptor. The pharmacological profile of the rat GALR2 receptor is similar to that of the rat GALR1 receptor. The rat GALR2 receptor binds galanin, N-terminal galanin fragments, and the putative galanin receptor antagonists galantide, C7, M35 and M40 with high affinity but it does not bind C-terminal galanin fragments. Galanin increases intracellular inositol phosphate levels in HEK 293 cells expressing the rat GALR2 receptor via a pertussis toxin-insensitive G-protein. The rat GALR2 receptor mRNA is highly expressed in several brain regions, including hypothalamus and hippocampus as well as the anterior pituitary, with lower levels of expression detected in amygdala, and regions of cortex. It is also highly expressed in the GH3 pituitary cell line and in gut tissues, and to a lower extent in spleen, lung, skeletal muscle, heart, kidney, liver and testis. These results suggest that GALR2 receptor mediates galanin's regulation of pituitary hormone secretion and possibly food intake.

Developmental regulation of two distinct neuronal phenotypes in rat dorsal root ganglia.

In a previous study we described two distinct neuronal phenotypes in rat dorsal root ganglia based on immunocytochemical assays for the neuronal intermediate filament proteins, peripherin and low-molecular-weight neurofilaments [Goldstein M. E. et al. (1991) J. Neurosci. Res. 30, 92-104]. In this paper we have extended this classification by using in situ hybridization to localize and evaluate the levels of various cytoskeletal and neuropeptide messenger RNAs within the peripherin-immunoreactive and peripherin-immunoreactive-negative neurons found in embryonic day 15 and 20, postnatal day 2 and adult dorsal root ganglia. We found in postnatal and adult dorsal root ganglia in vivo that the large, peripherin-immunoreactive-negative neurons, which are intensely stained by low-molecular-weight neurofilament antibodies, also contain high levels of low, medium and high-molecular-weight neurofilament messenger RNAs, whereas the smaller peripherin-immunoreactive neurons do not. On the other hand, both cell types contained comparable levels of peripherin and alpha-tubulin messenger RNA. The presence of peripherin messenger RNA but no peripherin immunoreactivity in the large cells suggested either a translational or post-translational regulation of this polypeptide, or rapid clearance of this protein from the perikaryon into the axon. In adult dorsal root ganglia, more than 50% of the peripherin-immunoreactive neurons also contained high levels of substance P and/or calcitonin gene-related peptide messenger RNAs, while less than 20% of the large peripherin-immunoreactive-negative neurons did. The attainment of these phenotypic characteristics during development in vivo was studied by northern blot and in situ hybridization histochemistry. In early embryonic stages (embryonic days 15-16), virtually all neurons were peripherin-immunoreactive and were positive for peripherin, alpha-tubulin and low-molecular-weight neuro-filament messenger RNAs, suggesting a homogeneous population. By embryonic day 20, the two adult phenotypes became clearly evident, and were fully established by postnatal day 2. In cultures of embryonic day 15 dorsal root ganglion neurons grown in the presence of nerve growth factor, peripherin and low-molecular-weight neurofilament messenger RNAs were expressed in all neurons, even after nine days in vitro, similar to embryonic dorsal root ganglia in vivo. Nerve growth factor supplemented by skeletal and heart muscle extracts did up-regulate neurofilament gene expression, but not to the extent characteristic of the peripherin-immunoreactive-negative adult phenotype. These results suggest that development of the mature phenotype of dorsal root ganglion neurons occurs by postnatal day 2 in vivo and is dependent upon target contact and/or target-derived factors.

Cloning and characterization of the rat GALR1 galanin receptor from Rin14B insulinoma cells.

Galanin is a ubiquitous neuropeptide that regulates a wide array of physiological processes via interaction with specific G protein-coupled receptors. A rat galanin receptor cDNA was cloned from the Rin14B insulinoma cell line. The isolated cDNA encodes a 346 amino acid G protein-coupled receptor that is 92% identical to the recently reported human GALR1 galanin receptor. [125I]Galanin binds with high affinity to two receptor states in COS1 cell membranes containing the rat GALR1 receptor, consistent with coupling of the receptor to a G protein in these membranes. N-terminal galanin fragments and the putative galanin receptor antagonists galantide, C7, M35 and M40 bind with high affinity to the rat GALR1 receptor. In contrast, C-terminal galanin fragments do not bind to this receptor. Galanin inhibits basal and forskolin-stimulated cAMP formation in CHO cells expressing the rat GALR1 receptor via a pertussis toxin-sensitive G protein. The GALR1 receptor is expressed in rat spinal cord, small intestine, Rin14B insulinoma cells and several brain regions, particularly ventral hippocampus, amygdala, supraoptic nucleus, hypothalamus, thalamus, lateral parabrachial nucleus and locus coeruleus. Cloning of the rat GALR1 galanin receptor cDNA will permit many new experimental strategies to be applied to studies of the structure and function of galanin receptors.

Neuronal sprouting in mouse sensory ganglia infected with herpes simplex virus type 2 (HSV-2): induction of growth-associated protein (GAP-43) and ultrastructural evidence.

Herpes simplex virus (HSV) is neurotropic and when inoculated on the mouse footpad is retrogradely transported to the associated dorsal root ganglia (DRG), where infection is established. Previous observations suggest that, after HSV infection, sensory ganglion neurons may mount a sprouting response. In our HSV-infected DRG model, we investigate this issue by (1) examining expression of growth-associated protein (GAP-43), a molecule known to be induced by growing axons, and (2) determining ultrastructurally whether HSV-infected dorsal roots contain neurites. In a time course study, we show that GAP-43 is induced both in HSV-infected DRG and their central processes. The increase in GAP-43 is first seen 2 weeks following unilateral footpad inoculation in both cell bodies and dorsal roots, and is sustained at 1 month post inoculation in roots but not in perikarya. Large bundles of unmyelinated small caliber axons, lacking Schwann cell ensheathment, are observed by electron microscopy in dorsal roots 2 weeks and 1 month following inoculation. These profiles resemble developing or regenerating neurites and are rarely seen in roots of mock-infected or uninfected controls. The increased GAP-43 immunoreactivity and ultrastructural changes shown here, in conjunction with previously documented selective neuropeptide and enzyme alterations, confirm that a sprouting response is mounted in sensory ganglia following acute HSV infection.

Peripheral deafferentation alters calcitonin gene-related peptide mRNA expression in visceral sensory neurons of the nodose and petrosal ganglia.

Visceral sensory neurons of the glossopharyngeal and vagus nerves are located in the petrosal and nodose ganglia, respectively. Our previous studies showed that peripheral axotomy which removes afferent input to visceral sensory perikarya decreased the number of calcitonin gene-related peptide (CGRP)-immunoreactive (ir) neurons in the petrosal but not the nodose ganglion. To evaluate axotomy-induced changes in CGRP mRNA expression, we used in situ hybridization histochemistry with 35S-labeled oligonucleotide probes. CGRP mRNA-containing neurons were studied 1, 3, 7 and 14 days after peripheral deafferentation of the left nodose and petrosal ganglia via transection of the left cervical vagus, superior laryngeal, glossopharyngeal and carotid sinus nerves. The numbers of CGRP mRNA-containing neurons in the deafferented petrosal ganglion were significantly reduced at 3, 7 and 14 days compared to either intact or sham-operated control ganglia. However, the density of hybridization product in the positively-labeled petrosal ganglion cells was not significantly changed. The numbers of CGRP mRNA-containing neurons in the deafferented nodose ganglion were significantly reduced at 3 and 7 days. These data suggest that axotomy-induced changes in CGRP-ir neurons of the petrosal ganglion correlate with changes in CGRP mRNA and probably result from altered CGRP gene expression. In addition, in situ hybridization histochemistry revealed changes in CGRP neurons of the nodose ganglion which were not apparent with immunocytochemistry.

Regulation of MHC class I and beta 2-microglobulin gene expression in human neuronal cells. Factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes.

MHC class I molecules are coexpressed with beta 2-microglobulin (beta 2-M) on many somatic cells. However, these proteins are normally not present on cells of the central nervous system (CNS). Cells derived from human neuroblastomas were used as a model for investigating the molecular basis for the paucity of MHC class I and beta 2-M gene expression in neural cells and for the induction of these genes by two cytokines, IFN-gamma, and TNF-alpha. These cytokines independently increased MHC class I and beta 2-M cell surface expression on the neuroblastoma cell lines. IFN-gamma or TNF-alpha also increased MHC class I and beta 2-M steady-state RNA levels and the expression of MHC class I and beta 2-M CAT reporter constructs transiently transfected into the neuroblastoma cell lines, indicating that the cytokines acted by increasing the transcription of these genes. MHC class I and beta 2-M genes share two conserved regulatory elements, an NF kappa B-like site and the IFN consensus sequence, that act as a constitutive enhancer and an IFN-responsive element, respectively. Low MHC class I and beta 2-M gene expression in these cells was accounted for by undetectable to low factor binding activity specific for the above regulatory elements of these genes. TNF-alpha increased factor binding activity specific for the NF kappa B-like elements and IFN-gamma increased factor binding activity specific for the IFN consensus sequence elements of the MHC class I and beta 2-M genes, but not vice versa. Taken together, our results indicated that IFN-gamma and TNF-alpha increased MHC class I and beta 2-M gene expression in the neuroblastoma cell lines by inducing factor binding to the regulatory elements present in both genes.

Herpes simplex virus type-2 infection by a footpad route results in neuronal death in mouse spinal ganglia.

We have examined the effects of herpes simplex virus type 2 (HSV-2) infection on neuron numbers in mouse dorsal root ganglia (DRG) following unilateral hind footpad inoculation. One month following HSV-2 strain MS inoculation, tissue sections of decalcified spine containing the paired 4th and 5th lumbar DRGs were stained with cresyl violet. Neuronal numbers, somal areas and ganglion volumes were determined for ganglia ipsilateral and contralateral to both HSV-2 and medium inoculations. One month following HSV-2 infection, 47-88% of neurons disappeared in the ipsilateral ganglia. The somal areas of the remaining neurons in these ganglia fell within the range of the uninfected population. Ganglionic shrinkage did not occur as a result of HSV-2 infection; neurons were replaced by large numbers of inflammatory cells. Neuron numbers in the contralateral control ganglia of the HSV-2 inoculated mice appeared slightly decreased. Mice inoculated with medium contained similar numbers of neurons in ipsilateral and contralateral ganglia. These results show that, in addition to other previously described host alterations, infection with HSV-2 strain MS results in neuronal death in the affected host ganglia. This is the first in vivo quantitative documentation of neuronal death induced by herpes simplex virus infection.

Molecular aspects of the regulation of tyrosine hydroxylase by testosterone.

Previous studies have demonstrated that the sympathetic hypogastric ganglia (HG) are dependent upon the continued presence of testosterone for normal development and maintenance of tyrosine hydroxylase (TH) activity. The regulation of TH by testosterone has been examined further to determine whether the reduction in TH activity following castration is associated with changes in levels of TH protein and mRNA. TH protein was measured by immunotitration of HG homogenates using a TH-specific antibody, and TH-specific mRNA was detected by hybridization of dot blots of total RNA isolated from HG with a cDNA probe coding for TH. The results show that tyrosine hydroxylase activity, protein and mRNA are coordinately reduced in a graded fashion at 1, 2 and 4 weeks following castration. Testosterone replacement therapy immediately following castration prevents the decrease in TH levels. The results indicate that gonadal steroids regulate the biosynthesis of TH in the HG. Testosterone may control TH either directly by interacting with neurons of the HG, or indirectly by altering levels of trophic factors in the target tissues.

NF-L and peripherin immunoreactivities define distinct classes of rat sensory ganglion cells.

Double immunofluorescence studies using antibodies against NF-L and peripherin revealed three distinct subpopulations of neurons in rat dorsal root ganglia (DRG). In the adult rat, 46% of the DRG neurons were small and peripherin-positive (NF-L-negative), and 48% were large and NF-L-positive (peripherin-negative). About 6% were both peripherin- and NF-L-positive. All of the DRG neurons reacted with antibodies to NF-M and nonphosphorylation-dependent or phosphorylation-independent antibodies to NF-H. The neuropeptides were predominantly found in the peripherin-positive small cell population. Eighty-seven percent of the peripherin-positive small cell population contained substance P immunoreactivity, while 43% of this cell population contained CGRP. In contrast, only 18-24% of the NF-L-positive large-cell population contained neuropeptides, and these were primarily in a smaller sized subpopulation. Similar patterns of antigen representation were observed in neonatal (PN2) DRG cell populations. Tissue cultures of sensory ganglion cells from PN2 DRG, in serum-free medium, stably maintained exclusively peripherin-positive neurons, with about 5% of these containing coexistent NF-L immunoreactivity. Very high levels of neuropeptide gene expression were exhibited by these postnatal neurons in culture.

mRNA levels of all three neurofilament proteins decline following nerve transection.

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.

Studies of neurofilaments that accumulate in proximal axons of rats intoxicated with beta,beta'-iminodipropionitrile (IDPN).

The paradigm of IDPN neuropathy was produced in rats in order to examine the neurofilaments (NFs) that accumulate in the proximal motor and sensory axons of intoxicated animals, and to compare the aggregated NFs with control NFs and with the depleted populations of NFs in the distal portions of the same experimental nerves. NFs were probed biochemically and histochemically, using a large and well-characterized library of monoclonal antibodies that included antibodies that are monospecific for each of the rat NF protein subunits (NF-H, NF-M, and NF-L) as well as antibodies that recognized differential phosphorylated states of rat NF-H and NF-M. All antibodies tested showed enhanced immunostaining of enlarged axons and of large spheroids in the spinal cord and dorsal root ganglia of experimental animals. Biochemical analyses of IDPN-treated animals revealed enrichment of NF-H, NF-M, and NF-L in homogenates of dorsal root ganglia and of proximal motor and sensory nerve roots as well as depletion of the three subunits in distal nerve roots and in sciatic nerves. Immunoblot revealed a uniform enrichment of NF-H, NF-M, and NF-L in NF aggregates as well as the same admixture of phosphorylated and dephosphorylated epitopes of NF-H and NF-M in experimental and in control tissues. The global increase of immunoreactivity in axonal swellings to antibodies that react with phosphorylated, nonphosphorylated, and phosphorylation-independent NF epitopes suggests that IDPN induces an accumulation of NFs in proximal axons without necessarily altering the state of NF phosphorylation.

Phosphorylation of neurofilament proteins and chromatolysis following transection of rat sciatic nerve.

States of phosphorylation of neurofilament proteins were examined in the perikarya of rat sensory and motor neurons between 3 and 28 d following either a distal transection [6-7 cm from the L4-L5 dorsal root ganglia (DRG)] or a proximal transection (1-2 cm from the L4-L5 DRG) of the sciatic nerve. Paraffin sections of the right (experimental) and left (control) L4 and L5 DRG from animals with unilateral transection of the right distal sciatic nerve were stained immunocytochemically with monoclonal antibodies to phosphorylation-dependent (NF-P), dephosphorylation-dependent (NF-dP), or phosphorylation-independent (NF-ind) epitopes on the largest (NF200), mid-sized (NF150), or smallest (NF68) neurofilament protein subunits. Increased immunoreactivity to NF-P on NF200 and NF150 was detected in experimental DRC at 10 d, peaking by 20 d, and declining to near control levels by 28 d. Conversely, immunoreactivity to NF-dP declined in experimental DRG beginning at 6 d, reaching a maximum decline at 10-16 d, and returning to near control levels by 28 d. Immunocytochemical changes were confirmed with biochemical studies on tissue homogenates that demonstrated an increase of immunoreactivity to NF-P and a decrease of reactivity to NF-dP in the experimental DRG. Changes in immunoreactivities to NF-P and NF-dP were observed only in the perikarya of large neurons and were closely associated with chromatolytic changes in these neurons. Marked enhancement of chromatolysis, as well as the immunoreactivities to NF-P and NF-dP, occurred following a proximal (left side) versus distal (right side) transection in the same animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation protects neurofilaments against proteolysis.

During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that phosphorylated neurofilaments are protected against proteolysis, and dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.

Varying degrees of phosphorylation determine microheterogeneity of the heavy neurofilament polypeptide (Nf-H).

Two-dimensional immunoblots revealed a spectrum of 200 kDa neurofilament polypeptides (Nf-H) of apparent molecular weights ranging from 200 to 170 kDa. The entire spectrum was stained immunocytochemically by three monoclonal antibodies specific for nonphosphorylated neurofilaments, while more restricted staining was revealed by four monoclonal antibodies specific for phosphorylated neurofilament epitopes. Treatment with increasing amounts of phosphatase suggested the existence of various forms of partially phosphorylated neurofilaments that possess phosphoepitopes that differ in their ease of dephosphorylation. Immunoprecipitation in low detergent concentration confirmed the existence of microheterogeneous forms of Nf-H that differed in extent of phosphorylation or in distribution of phosphorylated sites.

Microheterogeneity ("neurotypy") of neurofilament proteins.

Neurofilaments purified from adult rat brainstem by two methods were electrophoresed on NaDodSO4/polyacrylamide gels to separate the triplet proteins (approximate Mrs of 200,000, 155,000, and 68,000) which, in turn, were electroblotted onto nitrocellulose paper. On Coomassie blue-stained gels that were not electroblotted, the same banding pattern was seen with both methods of preparation. Immunocytochemical staining of the electroblots with each of five monoclonal antibodies revealed that three of the monoclonal antibodies were specific for the Mr 200,000 neurofilament protein and two, for both the Mrs 200,000 and 155,000 neurofilament proteins. None of the antibodies reacted with the Mr 68,000 band. The Mr 200,000 band could be resolved into doublet bands. Individual monoclonal antibodies reacted with either one or both of the Mr 200,000 doublets. The immunocytochemical staining of the neurofilament triplets on electroblots was compared to that of adult rat cerebellar paraffin sections. Each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes. Correlation of the staining patterns in cerebellar tissue sections with those of neurofilament polypeptides on electroblots suggested that different neurofilament polypeptides can be localized to different structures and subpopulations of neurons and that molecular heterogeneity ("neurotypy") may be revealed within the Mrs 200,000 and 155,000 neurofilament polypeptides.

Developmental expression of neurotypy revealed by immunocytochemistry with monoclonal antibodies.

Adult and developing rat cerebella were stained immunocytochemically with six neuron-specific monoclonal antibodies obtained from spleen cells of BALB/c mice immunized with hypothalamus. Monoclonal peroxidase-antiperoxidase complex was used in the staining procedure. In the adult, three different staining patterns were seen with these antibodies. The general patterns have been designated as (1) neurofibrillar, (2) perikaryal-neurofribillar, and (3) synapse-associated. In addition, each antibody within a designated group showed a different immunocytochemical distribution. Some antibodies reacted widely, for example, with many neuronal perikarya and fibers, and their distribution increased with the development of the brain. Other antibodies had a more restricted distribution and stained only some structures within an area or pathway. To account for this type of restriction of distribution, we propose that there may be microheterogeneity of some of the antigens visualized and have called this microheterogeneity 'neurotypy'. A second type of restriction was also observed. With several antibodies a loss of staining occurred in the adult cerebellum in structures that had reacted during early development. These differences in staining probably reflect developmental regulation of the antigens (neurotypes).