Immunocytochemical localization of protein kinase C isozymes in rat brain

Recently, we isolated 3 protein kinase C (PKC) isozymes from rat brain (Huang et al., 1986a). Using isozyme-specific antibodies for immunoblot, we have determined the relative levels of each isozyme in various regions of the rat brain (Huang et al., 1987b). The present paper describes the cellular distributions of PKC isozymes in rat brain as determined by light microscopic immunocytochemistry. Staining with PKC antibodies revealed strong immunoreactivities in neuronal somata and their dendrites and weak to no reaction in axon and the astroglial structures. In the cerebellum, the type I PKC antibodies stained the Purkinje cell bodies and dendrites; the type II PKC antibodies stained the granule cells; and the type III PKC antibody stained both Purkinje and granule cells. In the cerebral cortex, all antibodies stained neurons resembling pyramidal cells and their apical dendrites in layers II to VI, while layer I was nearly devoid of staining. However, the various isozyme-specific antibodies revealed distinct laminar distribution patterns of the positively stained neurons, and the type III PKC-positive neurons exhibited a higher density than those of type I or II PKC-positive ones, especially in layer II of cingulate (retrosplenial) and piriform cortices. In the hippocampal formation, both pyramidal cells of the hippocampus and granule cells of the dentate gyrus were stained by all PKC antibodies. Subcellularly, type III PKC appeared mostly in the cytoplasm of these neurons, whereas type I and II PKC seemed to associate with the nucleus as well. In the olfactory bulb, both type II and III PKC antibodies stained the periglomerular and granular cells, and the latter also stained the mitral cells. The distinct cellular and subcellular distribution of PKC isozymes suggests that each isozyme plays a unique role in the various neural functions.     

Neonatal Rat Cerebellar Granule and Purkinje Neurons in Culture Express Different GABAA Receptors

We have established a culture system for microexplants of rat cerebellar cortical tissue in which cells develop morphologically, express type-A receptors for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and form GABAergic synaptic connections. Criteria of cell size and shape allow reliable identification of granule and Purkinje neurons, criteria confirmed by studies of the binding of antibodies to calbindin D28K and GABA. Both granule and Purkinje neurons express GABA_A receptors, but granule neurons fall into two classes in terms of their sensitivity. Granule neurons which do not show spontaneous synaptic currents are relatively insensitive to GABA, while granule neurons with synaptic currents are much more sensitive. The responses of Purkinje neurons to applications of 1 μM GABA are relatively insensitive to Zn²⁺ ions (10 μM), and are potentiated by chlordiazepoxide (100 μM) and La³⁺ ions (100 μM). Responses of innervated granule neurons, on the other hand, are blocked more strongly by Zn²⁺ ions, are less affected by chlordiazepoxide and are equally potentiated by La³⁺ ions. Hence these cultures provide a source of identifiable, functionally innervated cells which express distinct types of GABA_A receptors.     

Developmental expression of protein kinase C isozymes in rat cerebellum

Previously we showed that protein kinase C (PKC) isozymes (types I, II, and III) have distinctive neuronal localizations in cerebellum. In the present study, we followed the different appearances of these isozymes during the postnatal development of cerebellum. By immunoblot analysis, type I PKC was found to be low within 2 weeks after birth; an abrupt increase was observed between 2 and 3 weeks and leveled off afterwards. By immunofluorescent staining, the type I PKC-specific antibody recognized the cell bodies and dendrites of Purkinje cells. The increase of this isozyme between 2 and 3 weeks of age correlates with the spreading of Purkinje cell arborization, at which time bulk of synaptogenesis between dendritic spines and axons of granule cells occurs. Both type II and III PKCs were present in granule cells. At birth, the level of type II PKC was relatively high compared to that of type III PKC, and the type II PKC-specific antibody stained the granule cell precursors in the external layer more heavily than did the type III PKC-specific antibody. The level of type II PKC declined slightly after birth and increased again at one week and plateaued after three weeks, whereas that of type III PKC increased gradually until leveling off after three weeks. Throughout the development, the type III PKC-specific antibody also stained the cell bodies of Purkinje cells but not their dendrites. These results demonstrate that the developmental expression of PKC isozymes is under separate control, and their distinct cellular and subcellular localizations suggest their unique functions in the cerebellum.     

Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons

Whole-cell voltage-clamp techniques were used to study the effects of the protein kinase C (PKC) activators phorbol esters and OAG on Ca and K currents in differentiated neurons acutely dissociated from adult hippocampus and in tissue-cultured neurons from fetal hippocampus. PKC activators had selective depressant effects on K currents, with persistent currents (IK and IK-Ca) being reduced and transient current (IA) being unaffected. In both cell types we recorded both high-voltage-activated, noninactivating (L-type) and high-voltage-activated, rapidly inactivating (N-type) Ca current. A low-voltage-activated, rapidly inactivating (T-type) Ca current was also recorded in tissue-cultured neurons but not in acutely dissociated neurons. PKC activators markedly reduced N-type current with less effect on L-type and no effect on T-type Ca current. Effects of PKC activators could be reversed with washing or with application of PKC inhibitors H-7 or polymyxin-B, an effect that could not be attributed to inhibition of cAMP-dependent protein kinase. The Ca/calmodulin inhibitor calmidazolium was ineffective in reversing the actions of PKC activators. Using whole-cell voltage-clamp techniques, we have demonstrated that hippocampal neurons possess 3 distinguishable components of calcium current. Distinct K currents were also observed. Our data strongly support the hypothesis that both Ca and K currents are selectively regulated by PKC and that these effects occur directly on the postsynaptic neuron.     

PLCgamma signaling underlies BDNF potentiation of Purkinje cell responses to GABA

Brain-derived neurotrophic factor (BDNF) regulates neuronal survival, neurite outgrowth, and excitatory synaptic transmission. We reported recently that acute BDNF exposure decreased gamma-aminobutyric acid (GABA) responses in cultured mouse cerebellar granule cells through tyrosine receptor kinase B (TrkB) receptor-mediated signaling. In the present study, we extend this work to investigate BDNF-induced modulation of GABA responses and GABA(A) receptor-mediated synaptic events in cerebellar slices. Thin (200 microm) parasagittal slices of cerebellum were prepared from postnatal Day 7 and 14 mice. Purkinje cells and granule cells, both of which express TrkB-like immunoreactivity, were identified for whole-cell recording. BDNF promptly enhanced GABA responses in Purkinje cells but, consistent with our previous finding in culture, attenuated those recorded in granule cells. In Purkinje cells, BDNF exposure shifted rightward the cumulative peak amplitude distribution for miniature inhibitory postsynaptic currents (mIPSCs) without changing the mIPSC frequency. BDNF-induced potentiation of Purkinje cell responses to GABA was blocked by TrkB-Fc (receptor body that sequesters BDNF), K252a (inhibitor of TrkB receptor autophosphorylation), U73122 (inhibitor of phospholipase-Cgamma [PLCgamma]), KN62 (specific inhibitor of calcium/calmodulin-dependent kinase), KT5720 (specific cyclic AMP-dependent kinase inhibitor), and by intracellular dialysis of Rp-cyclic AMP or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid). Overall, our results indicate that BDNF acutely potentiates GABA(A) receptor function in cerebellar Purkinje cells via the TrkB receptor-PLCgamma signal transduction cascade. In addition, we propose that cyclic AMP-mediated intracellular signaling mechanisms may facilitate manifestation of the BDNF-induced modulatory outcome.     

Synaptic organization of the mouse cerebellar cortex in organotypic slice cultures

The cellular and synaptic organization of new born mouse cerebellum maintained in organotypic slice cultures was investigated using immunohistochemical and patch-clamp recording approaches. The histological organization of the cultures shared many features with that observed in situ. Purkinje cells were generally arranged in a monolayer surrounded by a molecular-like neuropil made of Purkinje cell dendritic arborizations. Purkinje cell axons ran between clusters of small round cells identified as granule cells by Kv3.1b potassium channel immunolabelling. The terminal varicosities of the Purkinje cells axons enwrapped presumptive neurons of the cerebellar nuclei whereas their recurrent collaterals were in contact with Purkinje cells and other neurons. Granule cell axons established contacts with Purkinje cell somata and dendrites. Parvalbumin and glutamine acid decarboxylase (GAD) immunohistochemistry revealed the presence of presumptive interneurons throughout the culture. The endings of granule cell axons were observed to be in contact with these interneurons. Similarly, interneurons endings were seen close to Purkinje cells and granule cells. Whole cell recordings from Purkinje cell somata showed AMPA receptor-mediated spontaneous excitatory post-synaptic currents (sEPSCs) and GABAA receptor-mediated spontaneous inhibitory post-synaptic currents (sIPSCs). Similar events were recorded from granule cell somata except that in this neuronal type EPSPs have both a NMDA component and an AMPA component. In addition, pharmacological experiments demonstrated a GABAergic control of granule cell activity and a glutamatergic control of GABAergic neurons by granule cells. This study shows that a functional neuronal network is established in such organotypic cultures even in the absence of the two normal excitatory afferents, the mossy fibers and the climbing fibers.     

AMPA receptor subunit RNA transcripts and [(3)H]AMPA binding in the cerebellum of normal and pcd mutant mice: an in situ hybridization study combined with receptor autoradiography

The expression of AMPA receptor subunit mRNAs and the binding of [(3)H]AMPA were studied in the cerebellum of normal and "Purkinje cell degeneration" ( pcd) mutant mouse. In the pcd cerebellum, [(3)H]AMPA binding was decreased significantly in both the molecular and granule cell layers by 63% and 36%, respectively. In those mutants, GluRA, GluRB and GluRC mRNAs were not detected in the Purkinje cell layer, and the levels of GluRB and GluRD mRNAs were significantly decreased in the granule cell layer by 16% and 57%, respectively. Cerebellar grafts transplanted into the pcd cerebellum expressed only GluRB and GluRC mRNAs, suggesting that donor cells express the appropriate subunits normally expressed by Purkinje neurons. Our results, firstly, support the idea that the expression of the GluRA subunit in Golgi epithelial cells may depend upon the sustained interaction with adjacent Purkinje cells, and secondly, suggest that granule cells which are more resistant to transsynaptic death may express higher levels of GluRB mRNA.     

Modulatory Action of serotonin on glutamate-induced excitation of cerebellar purkinje cells

The effects of serotonin (5-hydroxytryptamine, 5-HT) on glutamate-induced excitation of Purkinje cells were examined. Pulsatile iontophoretic applications of glutamate (1-25 nA) induced consistent increases in the spontaneous activity of Purkinje cells. When serotonin was applied continuously with currents that elicited minimum changes in the spontaneous rate, it inhibited or blocked glutamate-induced excitations significantly in most Purkinje cells. We also examined the effects of high currents of serotonin on spontaneous activity of Purkinje cells. High currents of serotonin induced 3 different effects: inhibitions, biphasic effects comprising transient inhibition followed by excitation, and excitations. Nonetheless, whether it inhibited or excited the activity of Purkinje cells, serotonin inhibited glutamate-induced excitations consistently. The effect of a putative 5-HT antagonist methysergide (UML) was also examined. Methysergide consistently attenuated or antagonized the inhibitory effects of serotonin on glutamate-induced excitations. This finding suggests strongly that inhibitory effects of 5-HT on glutamate excitations observed in the present study is the specific action of serotonin.     

Protein Kinase C in the Cerebellum: Its Significance and Remaining Conundrums

Protein kinase C (PKC), a family of serine/threonine protein kinases, mediates a myriad of patho-physiological cellular events in various tissues. The originally discovered PKC (conventional) requires the binding of diacylglycerol and Ca²⁺ for full activation. The conventional PKC consists of four isoforms, PKCα, PKCβI/βII, and PKCγ. PKCα and PKCβI/βII are expressed in the cells of various tissues including the brain, while PKCγ is present specifically in neurons of the brain and spinal cord. The cerebellum expresses the largest amount of PKC with all its four isoforms. Purkinje cells express PKCα and PKCγ. Previous studies have shown that PKCα is involved in the induction of long-term depression (LTD) at parallel fiber–Purkinje cell synapses. On the other hand, analysis of PKCγ-deficient mice has revealed that PKCγ plays a critical role in eliminating supernumerary climbing fiber synapses from developing Purkinje cells. Although why PKCα has no compensatory action in climbing fiber pruning in PKCγ-deficient Purkinje cells had so far remained unclear, we have recently demonstrated that PKCα is also capable of pruning supernumerary climbing fiber synapses, but the expression levels of PKCα are too low to achieve pruning in PKCγ-null Purkinje cells. Notably, although PKCγ is most abundant in Purkinje cells, its physiological role in mature Purkinje cells remained totally unknown. In addition to a concise review of the physiological and pathological roles of conventional PKCs in Purkinje cells, this report postulates a contribution of PKCα in developing Purkinje cells and a possible involvement of PKCγ in motor coordination in the mature cerebellum.     

Evidence for hippocampal calcium channel regulation by PKC based on comparison of diacylglycerols and phorbol esters.

Studies using phorbol esters imply that hippocampal Ca2+ channels are regulated by protein kinase C (PKC); however concerns have been raised because in some circumstances phorbol esters have non-specific effects on ion channels. We have tested the hypothesis that PKC modulates Ca2+ channel activity in hippocampal neurons by conducting a detailed comparison of the effects of the diacylglycerols, diC8 and OAG, with those of the phorbol ester, PDBu, on whole-cell and single-channel Ca2+ currents. Close similarity of action of these different activators would support the hypothesis. We found that, like PDBu, the diacylglycerols (DAGs) suppressed whole-cell Ba2+ current (IBa) in a dose-dependent and reversible manner and caused a hyperpolarizing shift in the voltage dependence of steady-state IBa inactivation. Suppression of IBa by diC8 and OAG was not mimicked by an enzymatically inactive diacylglycerol isomer, EGD. The effects of both PDBu and DAGs could be blocked by a specific peptide inhibitor of PKC, and both types of activator depressed IBa when it was recorded in the nystatin perforated-patch mode. In single-channel recordings, DAGs enhanced L-type Ca2+ channel activity in a manner indistinguishable from that of PDBu. Finally, DAGs as well as PDBu markedly increased spontaneous synaptic activity in tissue-cultured hippocampal neurons. The numerous similarities between the effects of DAGs and PDBu strongly support the general conclusion that PKC mediates the effects of these activators and the specific conclusion that PKC modulates Ca2+ channel activity in hippocampal neurons.     

Neuronal migration defects in cerebellum of the<Emphasis Type="Italic">Large</Emphasis><Superscript>myd</Superscript> mouse are associated with disruptions in Bergmann glia organization and delayed migration of granule neurons

The Large gene encodes a putative glycosyltransferase that is required for normal glycosylation of dystroglycan, and defects in either Large or dystroglycan cause abnormal neuronal migration. The mechanism for this effect is not fully understood. This study analyzes the Largemyd mouse cerebellum during postnatal cerebellar development. Large is shown to be expressed most strongly in the Bergmann glial cells and Purkinje cells throughout cerebellar development, which is similar to what is known for dystroglycan expression. Discontinuities of the pial surface of the developing Largemyd mouse cerebellum correlate with disruption of the normal organization of the external granule cell layer and Bergmann glial fibers. At early time points, granule neurons express differentiation markers normally, both temporally and spatially, and show no defects in neurite outgrowth in in vitro assays. However, granule neuron migration is delayed within the external granule and molecular layers, resulting in granule neurons undergoing their intrinsically programmed differentiation in inappropriate locations. Consequently, cells expressing mature granule neuron markers become stranded within these layers. The cause of the less efficient migration is likely due to both physical disruption of the glial-guide scaffolding, as well as to suboptimal neuronal-glial guide interactions during migration.     

Granule cell raphes in the cerebellar cortex of chicken and mouse

The cerebellar cortex of the chicken embryo contains parasagittal segments of Purkinje cells. At intermediate stages of development, cell-dense ribbons of migrating granule cells ("raphes") are found between the segments. The complementary pattern of granule cell raphes and Purkinje cell segments represents a basic scheme of cerebellar organization that coincides with the expression domains of various genes, such as cadherins, gene regulatory proteins, and ephrins and their receptors. We have recently found the raphe/segment pattern also in a mammalian species, the postnatal mouse. Like in the chicken, the parasagittal raphes of granule cells were observed at the boundaries of Purkinje cell segments that differentially express cadherins. The number and arrangement of the raphes in the different cerebellar lobules is roughly similar in both species. The raphe/segment pattern is thus more widely distributed in vertebrates than previously assumed.     

Characterization of an antiserum to synaptic glomeruli from rat cerebellum

Rabbit anti-rat cerebellar synaptic glomeruli antiserum when absorbed with non-neural tissues reacts only with neural tissues when tested by indirect immunofluorescence on tissue sections. Further absorption with forebrain results in an antiserum which detectably reacts only with synaptic glomeruli and soma of Purkinje cells of both rat and mouse. The developmental expression of the synaptic glomeruli antigen(s) parallels the formation of synapses between mossy fibers and granule cells. Immature synaptic contacts do not contain recognizable antigent(s), whereas only at postnatal Day 15 glomeruli become antigen-positive. At this stage antigen in Purkinje cells is no longer carried in their dendrites, but becomes confined to the cell soma. Staggerer mutant mice still express the immature pattern of antigen distribution on postnatal Day 18.     

Immunohistochemical analyses of DNA topoisomerase II isoforms in developing rat cerebellum

In mammalian cells, two isoforms of DNA topoisomerase II (topo IIalpha and topo IIbeta) have been identified. Topo IIalpha is essential in mitotic cells, whereas the function of topo IIbeta remains unclear. In the present study, we investigated the developmental control of topo II isoforms in two different neuronal lineages, cerebellar Purkinje cells and granule cells, by immunohistochemical analysis with isoform-specific monoclonal antibodies. As expected, proliferating cells in the neuroepithelium and in the external germinal layer (EGL) were topo IIalpha immunopositive. The migrating as well as differentiating Purkinje cells and granule cells showed an enhanced topo IIbeta immunoreactivity. The postmitotic granule cells in the postnatal EGL showed an abrupt transition of expressed topo II isoforms from IIalpha to IIbeta. The transition was clearly coincident with the completion of final cell division and the initiation of terminal differentiation because no increase of the topo IIbeta immunoreactivity was observed in the spreading EGL cells that are still in the cell division cycle. The topo IIbeta signal was detected in both nucleoplasm and nucleolus of differentiating cells. However, the nucleoplasmic signal decreased significantly as the cells reached terminal differentiation. The residual topo IIbeta in nucleoli was shown to occupy an unique location with respect to other nucleolar proteins, nucleolin and DNA topoisomerase I. Our findings indicate that both Purkinje cells and granule cells express the topo II isoforms in a similar timing during the cerebellar development and also suggest that topo IIbeta localized in nucleoplasm is the functional entity involved in neuronal differentiation.     

Granule cells migrate within raphes in the developing cerebellum: an evolutionarily conserved morphogenic event.

The early phase of granule cell migration in the developing chick cerebellum occurs within ribbons of cells moving through parasagittally arrayed gaps between Purkinje cell clusters. These parasagittal arrays of migrating granule cells, termed "granule cell raphes," also have been reported in rabbit and cat, but recent publications variously report that granule cell raphes are absent or present in rodents. By using Nissl counterstaining and Pax6 immunohistochemistry, we confirm that granule cells do migrate in raphes in the developing mouse cerebellum, and also in the primate cerebellum during a period of development that coincides with Purkinje cell compartmentation. In mouse and primate cerebellum, as in chick cerebellum, granule cell migratory streams occur at the borders of Purkinje cell clusters. GFAP immunostaining of Bergmann glial fibers shows no parasagittally localized pattern of distribution, indicating that the formation of granule cell ribbons is not prepatterned by heterogeneous distribution of radial glia. The conservation of the ribboned pattern of granule cell migration from bird to primate and the timing of this event suggest a possible role for granule cell raphes in parasagittal compartmentation of Purkinje cells. A potential mechanism for such an interaction is discussed.     

Abnormalities of foliation and neuronal position in the cerebellum of NZB/BINJ mouse.

To analyze developmental abnormalities related to neural migration in the NZB/BINJ mouse, the pattern of cerebellar foliation and neural position were compared with that of a normal mouse (C57BL/6J). Three abnormalities of cerebellar foliation--(1) lobe isolated from other cerebellar lobes, (2) lobes imbalanced in relative amounts or ratio of granular cell layer and molecular layer, (3) lobes in which some Purkinje cells and the molecular layer was embedded in the granular cell layer--were observed in NZB/BINJ mice. These morphological abnormalities were not limited to a specific lobe. On the other hand, abnormalities of neural position were observed in both granule and Purkinje cells. The pattern of ectopically-situated granule cells, in general, could be divided into 3 types: (1) large cell clusters extending from granular cell layer to the pia mater or middle part of the molecular layer, (2) clusters of various sizes scattered within the white matter and (3) clusters formed by combination of granule cells extending from two opposed granular cell layers to the molecular layer. The pattern of ectopically-situated Purkinje cells could be divided into 4 types: (1) ectopia of a group of cells from one part of the Purkinje cell layer, (2) ectopia of a single Purkinje cell observed in the molecular layer, (3) single Purkinje cell scattered within the white matter accompanied by clusters of ectopic granule cells and (4) ectopic Purkinje cells embedded in the granular cell layer. The abnormalities in position of both granule cells and Purkinje cells was not limited to a particular cerebellar lobe.(ABSTRACT TRUNCATED AT 250 WORDS)     

The localization of GABAA receptors in mice with mutations affecting the structure and connectivity of the cerebellum

The distribution of cerebellar [3H]muscimol binding sites was studied autoradiographically in normal C57BL/6J mice and in the weaver, reeler, Purkinje cell degeneration and staggerer mutant mice. In the normal 79-day-old mouse cerebellum, the highest concentration of [3H]muscimol binding sites was observed in the granule cell layer. A much lower grain density was present over the Purkinje cell and molecular layers and negligible numbers of binding sites were seen over the deep cerebellar nuclei and white matter. A significant decrease in [3H]muscimol labeling was observed over the cerebellar cortex of the 81-86-day-old weaver mutant; this was most pronounced in the vermis where granule cell loss was the greatest. Over the hemispheres, where fewer granule cells degenerate, a higher density of binding sites remained. In the 27-29-old reeler cerebellum, where Purkinje cells are malpositioned, no labeling was seen over the deep Purkinje cell masses. In the quasi-normal superficial cortex, labeling density over the surviving granule cell layer was only slightly decreased. In the 54-57-day-old Purkinje cell degeneration mutant, where essentially all Purkinje cells have disappeared by day 45, a 29% decrease in grain density over the granule cell layer was observed, while labeling was still present in the molecular layer. Virtually no [3H]muscimol labeling was detected over any part of the cerebellar cortex of the 25-27-day-old staggerer mutant (which lacks parallel fiber-Purkinje cell synapses), although clusters of surviving granule cells were present in significant numbers in the lateral aspects of the cortex. Our autoradiographic data indicate that GABAA receptors are associated with granule cells in both the molecular and granule cell layers. Furthermore, our results raise the possibility that the maintenance of receptor levels may be dependent upon synaptic contacts between the granule cell and its main postsynaptic target, the Purkinje cell.