Richard Hawkes

Monoclonal antibodies reveal sagittal banding in the rodent cerebellar cortex

We have produced two monoclonal antibodies against polypeptide-associated antigens of developing rat cerebellum. One antibody recognizes an antigen associated with synaptic vesicles and another binds to a polypeptide which is restricted to the cytoplasm of a subset of cerebellar Purkinje cells. Both antibodies reveal the biochemical differentiation of the rodent cerebellar cortex into antigenically distinct sagittal zones.

The compartmentalization of the monkey and rat cerebellar cortex: zebrin I and cytochrome oxidase

The cerebellar cortex of mammals is composed of parasagittal zones that encompass the afferent inputs, the efferent corticonuclear and corticovestibular projections, and a number of intrinsic molecular markers. One such marker is the polypeptide antigen zebrin I that is recognized by monoclonal antibody (mab) Q113. In rodents, zebrin I immunocytochemistry reveals an array of parasagittal Purkinje cell compartments. In the present study, zebrin I has been used to reveal the molecular heterogeneity of the cerebellar cortex in the squirrel monkey (Saimiri sciureus). As in rodents, zebrin I is Purkinje cell specific in the primate cerebellum and not all Purkinje cells are immunoreactive. Immunocytochemistry on frontal or horizontal sections reveals a system of bands of zebrin I+ cells extending through the vermis of both anterior and posterior lobes. A midline (P1+) band and two more lateral bands (P2+ and P3+) are found in all lobules. The situation in the paravermis and hemispheres is similar, with alternating zebrin I+ and zebrin I- compartments, but the complex lobulation obscures the precise band pattern: it seems probable that 4 additional bands are present in the hemispheres, as in rodents. Comparison of rat and monkey cerebellums suggests that the cortex has expanded in primates by the growth of the same individual bands found in rats rather than by the addition of supplementary compartments. The zebrin I compartmentalization revealed by using mab Q113 is reproducible from individual and thus provides a stable frame of reference that has been used to compare the different chemoarchitectonic patterns found in the cerebellar cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Zonation in the rat cerebellar cortex: patches of high acetylcholinesterase activity in the granular layer are congruent with Purkinje cell compartments.

The rat cerebellar cortex is built from parasagittally arranged modules with topographically ordered afferent and efferent projections. The intrinsic organization of the cerebellum is revealed by immunocytochemical staining with monoclonal antibody, mabQ113. In the cerebellum, mabQ113 recognizes a polypeptide epitope that is restricted to a subset of Purkinje cells. Antigenic Purkinje cells are clustered to form a complex pattern of parasagittal compartments. Several biochemical markers reveal a superficially similar organization of the cortex, and so it is important to determine how many independent maps are present. This report compares the mabQ113 antigen display to the patchy distribution of acetylcholinesterase (AChE). In the granular layer and the white matter of the adult cerebellar cortex there is a patchy AChE staining that includes both the hemispheres and the vermis. The staining is often not sharply resolved cytologically, but seems to be associated primarily with the synaptic glomeruli. The boundaries of these granular layer patches in the vermis correspond to the mabQ113+/mabQ113- boundaries of the overlying Purkinje cell compartments. Thus, AChE and mabQ113 antigen share a common compartmentation both in the vermis, and in the hemispheres. Both mabQ113 and AChE distributions develop postnatally in the cerebellar cortex. At birth (PO) there is neither AChE activity nor mabQ113 immunoreactivity. Both staining patterns emerge during the second postnatal week. In the vermis at P10, there is AChE activity in the granular layer and white matter, and the distribution is already patchy despite the absence of synaptic glomeruli. At the same age the mabQ113 immunoreactivity is found in all Purkinje cells rather than a subset, and the band pattern has yet to mature. There is also transient AChE staining of Purkinje cell somata and dendrites. The AChE patches clarify between P10 and P20 along with the appearance of the synaptic glomeruli and the development of differential mabQ113 staining, but there is no reason to believe that the two are causally linked. In contrast to the cerebellar cortex, AChE staining in the cerebellar nuclei matures very early and at P0 the activity is already high. Zones of high and low AChE activity are seen in all the cerebellar nuclei and may be related to the distribution of the terminal fields of the different Purkinje cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Purkinje cell axon collateral distribution reflect the chemical compartmentation of the rat cerebellar cortex

Monoclonal antibody mabQ113 has been used to study the distribution of Purkinje cell axon collaterals in the rat cerebellar cortex. MabQ113 recognizes a polypeptide antigen, zebrin I, that is confined to a subset of Purkinje cells. Antigenic Purkinje cells are arranged in parasagittal compartments running throughout the cortex. No other cerebellar cells are immunoreactive. Immunoreactive axon collaterals are confined principally to the infraganglionic plexus with only a few extending into the molecular layer. Three probable target cells for the axon collaterals have been identified: Golgi cells, Lugaro cells, and other Purkinje cells. About 90% of immunoreactive axon collaterals in the anterior lobe are located beneath the mabQ113+ Purkinje cell compartment in which they originate but some do invade the neighboring mabQ113- territory. In the anterior lobe vermis, the distribution of invading mabQ113+ collaterals is not symmetrical, such that the probability of an invading collateral from the P2+ compartment is greater at the medial boundary into P1- than at the lateral into P2-. The distribution of immunoreactive collaterals is consistent with their playing a role in synchronizing the firing of Purkinje cells within the same compartment.

Characterization of novel glycoprotein components of synaptic membranes and postsynaptic densities, gp65 and gp55, with a monoclonal antibody.

A monoclonal antibody, mab SMgp65, which recognises two major glycoprotein components of isolated forebrain synaptic subfractions has been raised. The mab has been used to study the cellular and subcellular localisation of these novel glycoproteins and for the partial characterisation of both molecular species. Western blots show that the mab reacts with two diffuse glycoprotein bands (gp) of apparent Mr 65,000, gp65, and 55,000, gp55. Both glycoproteins are membrane-bound, only detectable in CNS tissue and exist solely in a concanavalin A (con A) binding form. Digestion with endoglycosidase H lowers the Mr of both glycoproteins by some 5-7 kDa. Gp65 and gp55 are enriched in synaptic membrane (SM), light membrane (LM) and microsomal fractions. However, whilst gp65 is enriched in isolated postsynaptic densities (psds) gp55 is conspicuously absent from this fraction. Regional distribution studies show a marked variation in the level of gp65. Gp65 is concentrated in several forebrain regions notably cerebral cortex, hippocampus and striatum, is present only in low levels in cerebellum and is barely detectable in pons and medulla. In contrast gp55 is present in all regions studied, but is most concentrated in cerebellum. Immunocytochemical studies show intense staining of regions rich in gp65, but no staining of regions deficient in this glycoprotein. This suggests that the mab recognises gp65, but not gp55 in fixed tissue sections. Exposure of tissue sections to Triton X-100 increases the intensity of gp65-like immunoreactivity, but does not alter its pattern of subcellular distribution. Higher resolution studies show the immunoreactivity to be localised to subsets of neurites, many being axonal. The reaction deposits also extend into the synaptic region of the immunoreactive neurones. Cultured cerebellar granule cells, but not astrocytes express gp55. The results are discussed in terms of the molecular properties and localisation of these two novel glycoproteins.

Selective staining of a subset of Purkinje cells in the human cerebellum with monoclonal antibody mabQ113

MabQ113 is a monoclonal antibody raised against rat cerebellum which selectively strains Purkinje cells. Likewise, in mabQ113-immunoperoxidase stained sections of human cerebellum, deposits of reaction product are found only in the Purkinje cells. The dendritic arborizations, cell body, and axonal processes are immunoreactive. In rat, mabQ113 reveals a series of parasagittal antigenic bands which run throughout the cerebellar cortex. The staining distribution in human cerebellar cortex likewise reveals heterogenous staining but the pattern is a complex one and seems to be unlike the parasagittal banding found in the rat. In a number of human diseases Purkinje cell degeneration is not uniform throughout the vermis and cerebellar hemispheres. It is possible that mabQ113+ and mabQ113- subsets of Purkinje cells may respond differentially to various pathological conditions.

Focal axonal swellings in rat cerebellar Purkinje cells during normal development

Focal axonal swellings are characteristic of a wide range of neuropathies. Three neuron-specific monoclonal antibodies have been used to identify focal axonal swellings in the normal developing rat cerebellar cortex. Between 7 and 15 days postnatal, swellings are a common feature of the granular layer and white matter tracts. Using a Purkinje cell-specific antibody, the majority of swellings were shown to occur in Purkinje cell axons. Focal axonal swellings therefore seem to be a normal adjunct of Purkinje cell maturation.

Thyroid hormone modulates the expression of a neurofilament antigen in the cerebellar cortex: premature induction and overexpression by basket cells in hyperthyroidism and a critical period for the correction of hypothyroidism

Neurofilament expression by basket cells of the cerebellar cortex is suppressed in hypothyroidism. By using a monoclonal antibody (mabN210) that selectively recognizes an epitope associated with the 210-kDa neurofilament subunit, we have explored the relationship between thyroid hormone levels and basket cell maturation. In animals rendered hypothyroid by inclusion of propylthiouracil in the maternal drinking water from embryo age E17, there is a complete absence of mabN210 immunoreactivity in the basket cell axons, while the other immunoreactive axons in the cerebellar cortex, primarily Purkinje cell axons and mossy fibers, are apparently unaffected. This deficit can be corrected by treatment with thyroid hormone but there seems to be a critical period for full recovery, for animals treated from birth recover normally whereas there is a gradual diminution in the efficacy of treatment the later it begins. Thyroid hormone therapy begun after postnatal day 30 (P30) leads only to very minor recovery. By contrast, animals on a hyperthyroid regime show premature mabN210-antigen induction in the basket cells and supranormal levels of expression at P25, despite the severe reduction in the number of basket cell somata. This suggests either abnormal compensatory sprouting of axon collaterals by the remaining basket cells or the occurrence, during normal cerebellar corticogenesis, of competition between basket cell axons for a limited number of Purkinje cell targets followed by the elimination of the excess collaterals.

Monoclonal antibodies reveal the global organization of the cerebellar cortex

Electrophysiological mapping of the rat cerebellar cortex has revealed an elaborate functional somatotopy that tract tracing procedures have shown to correlate with specific patterns of afferent and efferent connectivity that encompass the cerebellum as a whole. In contrast, most anatomical and biochemical procedures suggest that the cerebellar cortex is remarkably uniform. To unmask covert molecular heterogeneity underlying the functional map, it is appropriate to use monoclonal antibody technology to search for antigenic epitopes whose cerebellar distribution reflects or encodes the positional information. Given that no preconditions can be set on the biochemical nature of the putative epitopes, a shotgun approach to immunization and screening is required. The construction of monoclonal antibodies and screening for specificities that reveal positional information is discussed with examples from an anti-cerebellar antibody library.

Selective suppression of neurofilament antigen expression in the hypothyroid rat cerebral cortex

As an integral component of the cytoskeleton neurofilaments play a central role in the establishment and maintenance of neuronal form. In particular, high neurofilament concentrations are characteristic of many classes of axons in the central nervous system. Isolated neurofilaments from rat brain consist of 3 distinct polypeptides with apparent molecular weights 210K, 160K and 68K. A murine monoclonal antibody, mabN210, has been produced which specifically recognizes an epitope associated with the high molecular weight subunit and this antibody has been used to explore the regulation of neurofilament expression during brain development. It has been shown that in the rat cerebellar cortex, the expression of mabN210-immunoreactivity in basket cell axons is severely suppressed in hypothyroidism while neurofilament antigen expression in other cerebellar axons seems not to require thyroid hormones. In view of the well-known cortical deficits in hypothyroidism, these studies have now been extended to include the developing rat cerebral cortex and selected cortical afferent and efferent axons. In hypothyroid rats there is a marked suppression of mabN210-immunoreactivity in the cerebral cortex and corpus callosum and, to a lesser extent, there is a reduction in staining in the internal capsule. By contrast, hypothyroidism did not reduce mabN210-immunoreactivity in the lateral olfactory tract or the stria medullaris. In rats, serum thyroid hormone starts to rise to adult levels on postnatal day 4. It appears that axons that have attained their mature distribution prior to the onset of thyroid hormone expression are not affected by hypothyroidism whereas mabN210-immunoreactivity is suppressed in those axonal tracts that reach a mature distribution after P4.