Kenneth G. Baimbridge

Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat

The immunohistochemical localization of calcium-binding protein (CaBP) in the cerebellum, hippocampal formation and olfactory bulb of the rat was examined using rabbit anti-human or sheep anti-chick antisera purified by affinity chromatography. CaBP-like immunoreactivity was observed within the somata and dendrites of: (1) cerebellar Purkinje cells; (2) dentate granule cells, CA1 pyramidal cells and scattered interneurons in the stratum radiatum of the hippocampus; (3) periglomerular cells in the olfactory bulb. Staining was conspicuously absent in certain major cell types in each of these structures including cerebellar granule cells, hippocampal pyramidal cells in the CA3 region and both mitral and granule cells in the olfactory bulb. Immunoreactive fibers in the cerebellum presumably corresponding to climbing fiber inputs from the inferior olive and efferent projections to the deep cerebellar nuclei, were also observed. In the hippocampus intense staining was present in the mossy fiber projection to the CA3 region and in the terminal regions of the perforant path projection from entorhinal cortex.

Calcium-binding protein distribution in the rat brain

The distribution in the rat brain of a protein with properties similar to vitamin D-dependent calcium binding protein (CaBP) was measured using a radioimmunoassay developed for mammalian brain CaBP. The cerebellum contained very high levels of CaBP in agreement with reports of CaBP distribution in the chick brain. The protein was distributed in large and unequal concentrations in different regions of the brain. Microdissection of the hippocampus revealed that, within a particular region, CaBP can have a marked differential distribution, suggesting that it is found in specific cell types. The significance of this protein is discussed in relation to the important functions of calcium in the CNS with particular emphasis being placed upon the possible role of CaBP as an intraneuronal calcium ion buffering system.

Relative sparing in Parkinson's disease of substantia nigra dopamine neurons containing calbindin-D28K

The distribution of calbindin-D28K (CaBP)-positive neurons was investigated by immunohistochemistry in 4 controls, 5 cases of Parkinsons disease and a single case of strionigral degeneration. CaBP-positive neurons were preferentially localized to the mediodorsal portion of the substantia nigra pars compacta (SNC) in the beta layer, while CaBP-negative, melanin-positive neurons were concentrated in the ventrolateral SNC in the alpha layer. In Parkinsons disease and the case of strionigral degeneration, there was a relative sparing of the CaBP-positive neurons compared with CaBP-negative, pigmented neurons. These data imply that CaBP may confer some protection to SNC dopaminergic neurons against the pathological process which is responsible for Parkinsons disease and strionigral degeneration.

Relationship of neuronal vulnerability and calcium binding protein immunoreactivity in ischemia

SummaryThe relationship between neuronal calcium binding protein content (calbindin D28K: CaBP and parvalbumin : PV) and vulnerability to ischemia was studied in different regions of the rat brain using the four vessel occlusion model of complete forebrain ischemia. The areas studied, i.e. the hippocampal formation, neocortex, neostriatum and reticular thalamic nucleus (RTN), show a characteristic pattern of CaBP and PV distribution, and are involved in ischemic damage to different degrees. In the hippocampal formation CaBP is present in dentate granule cells and in a subpopulation of the CA1 pyramidal cells, the latter being the most and the former the least vulnerable to ischemia. Non-pyramidal cells containing CaBP in these regions survive ischemia, whereas PV-containing non-pyramidal cells in the CA1 region are occasionally lost. Hilar somatostatin-containing cells and CA3 pyramidal cells contain neither PV nor CaBP. Nevertheless, the latter are resistant to ischemia and the former is the first population of cells that undergoes degeneration. Supragranular pyramidal neurons containing CaBP are the most vulnerable cell group in the sensory neocortex. In the RTN the degenerating neurons contain both PV and CaBP. In the neostriatum, ischemic damage involves both CaBP-positive and negative medium spiny neurons, although the degeneration always starts in the dorsolateral neostriatum containing relatively few CaBP-positive cells. The giant cholinergic interneurons of the striatum contain neither CaBP nor PV, and they are the most resistant cell type in this area. These examples suggest the lack of a consistent and systematic relationship between neuronal CaBP or PV content and ischemic vulnerability. It appears that some populations of cells containing CaBP or PV are more predisposed to ischemic cell death than neurons lacking these proteins. These neurons may express high levels of calcium binding proteins because their normal activity may involve a high rate of calcium uptake and/or intraneuronal release.

Calretinin is present in non-pyramidal cells of the rat hippocampus—II. Co-existence with other calcium binding proteins and gaba

The possible co-existence of calretinin with other calcium binding proteins, parvalbumin and calbindin D28k, and with GABA, was studied in non-pyramidal cells of the rat dorsal hippocampal formation, using the mirror technique. The majority of the calretinin-containing neurons (83%) were found to be immunoreactive for GABA (79% in the dentate gyrus, 84% in the CA2-3, and 88% in the CA1 subfield). Most of the GABA-negative calretinin-immunoreactive neurons were located in the hilus of the dentate gyrus and in stratum lucidum of the CA3 subfield. Detailed analysis of the calretinin-immunoreactive cells of these subfields revealed that the two morphologically distinct types of calretinin neurons, i.e. the spiny and the spine-free cells, differ in their immunoreactivity for GABA. The overwhelming majority (92%) of the spine-free neurons were GABA-positive, whereas the immunoreactivity of spiny cells was ambiguous. At the sensitivity threshold of the immunocytochemical techniques used in the present study, most of the spiny cells (89%) had to be considered as GABA-negative, although the staining intensity in their cell bodies was somewhat above background level. Colchicine treatment resulted in a degeneration of calretinin-immunoreactive neurons; therefore, its effect on the GABA content of spiny neurons could not be evaluated. Nevertheless, the observations suggest that calretinin-containing neurons are heterogeneous both morphologically and neurochemically. Examination of the co-existence of calcium binding proteins revealed that none of the hippocampal cells contained both calretinin and parvalbumin in any regions of the hippocampal formation. Some overlap was detected between the calretinin- and the calbindin D28k-containing cell populations, 5.1% of the former and 6.2% of the latter were immunoreactive for both calcium binding proteins. This may be due to a small degree of cross-reactivity of the calbindin D28k antiserum with calretinin. Thus, our results demonstrate that the majority of calretinin-immunoreactive neurons are GABAergic and represent a subpopulation of non-pyramidal cells with no or only a negligible overlap with the subpopulations containing the other calcium binding proteins, parvalbumin and calbindin.

Potentiation of NMDA receptor-mediated excitotoxicity linked with intrinsic apoptotic pathway in YAC transgenic mouse model of Huntington's disease.

Evidence suggests N-methyl-D-aspartate receptor (NMDAR) activation is involved in the degeneration of striatal medium-sized spiny neurons (MSNs) in Huntingtons disease (HD). We tested the hypothesis that enhanced NMDAR-mediated excitotoxicity is mediated by the mitochondrial-associated apoptotic pathway in cultured MSNs from YAC transgenic mice expressing full-length huntingtin (htt) with a polyglutamine (polyQ) expansion of 46 or 72 (YAC46 or YAC72). NMDAR-mediated Ca(2+) transients and mitochondrial membrane depolarization were significantly increased in YAC compared to wild-type mice MSNs. Inhibitors of the mitochondrial permeability transition (mPT), cyclosporin A and bongkrekic acid, and coenzyme Q10 (an anti-oxidant involved in bioenergetic metabolism) dramatically diminished NMDA-induced cell death and eliminated genotypic differences. In YAC46 MSNs, NMDA stimulated significantly higher activation of caspase-3 and caspase-9 but not caspase-8, and NMDA-induced caspase-3 and -9 activation was markedly attenuated by cyclosporin A. Agents that improve mitochondrial function or inhibit the permeability transition may eliminate increased caspase activation and cell death associated with enhanced NMDAR activity in HD.

Calcium-induced long-term potentiation in the hippocampus.

The effect of a transient increase in extracellular calcium concentration on the Schaffer collateral-commissural evoked excitatory postsynaptic potential and population spike responses of CAI pyramidal neurons was investigated using the rat in vitro hippocampal slice preparation. Brief exposure of slices (5-10 min) to twice the normal concentration of calcium (4 mM) induced a marked potentiation of both the excitatory postsynaptic potential and population spike that could persist for at least 3 h. No long-term changes were observed in either the presynaptic fiber volley of antidromically evoked CAI population spike, indicating that the potentiation could not be attributed to an increase in the number of fibers activated or a generalized increase in cellular excitability. The response of CAI pyramidal neurons to the iontophoretic application of L-glutamate in the apical dendritic zone was also unaffected after exposure to high calcium perfusate, suggesting a lack of alteration in membrane excitability or receptor sensitivity restricted to the region of synaptic input. In addition, total intracellular calcium content of individual slices, measured by atomic absorption spectrophotometry, was significantly increased for at least 1 h following return to the control medium. These data indicate that brief exposure of in vitro hippocampal slices to a high extracellular calcium concentration results in a long-term increase in synaptic efficacy which is similar in many respects to long-term potentiation induced by tetanic stimulation of hippocampal excitatory afferents. The results further suggest that the mechanisms underlying calcium-induced long-term potentiation may reside in presynaptic components and involve an enhanced transmitter release.

Topographical localization of neurons containing parvalbumin and choline acetyltransferase in the medial septum-diagonal band region of the rat

The normal morphology and distribution of parvalbumin-containing neurons (shown in a previous study to be GABAergic nerve cells) of the medial septal-diagonal band region of the adult rat brain have been studied, and the findings compared with observations on choline acetyltransferase-immunoreactive neurons. The two antigens were visualized either in the same sections using a double-label immunohistochemical procedure for the simultaneous localization of parvalbumin and choline acetyltransferase, or in immediately adjacent sections. In double-stained sections of the whole medial septal-diagonal band complex, about 34% of the total neurons showed immunoreactivity to parvalbumin; the proportion of parvalbumin-labelled neurons was slightly higher in the medial septal-vertical limb of the diagonal band region, and much lower in the horizontal limb of the diagonal band region. The distribution of parvalbumin- and choline acetyltransferase-containing neurons also varied markedly between different mediolateral subdivisions of the medial septum: about 30, 65 and 2% of the parvalbumin-immunoreactive neurons were present in the midline, medial and lateral part of the medial septum, respectively. At different rostrocaudal levels, the proportion of parvalbumin- and choline acetyltransferase-positive neurons varied in a consistent manner, and the largest number of parvalbumin-containing neurons was found at the level 1.9 mm anterior to the bregma. In the absence of reliable immunocytochemical methods for the localization of glutamate decarboxylase and GABA, parvalbumin may serve as a good marker for studying the distribution of GABAergic neurons in the medial septum-diagonal band region. Moreover, the precise maps reported in the present study of the topographic localization of parvalbumin-containing GABAergic and choline acetyltransferase-immunoreactive cholinergic nerve cells in the medial septal-diagonal band complex will serve as a useful guide in future morphological and electrophysiological studies on the septum and its efferents.

Dantrolene-Na(Dantrium) blocks induction of long-term potentiation in hippocampal slices

Long-term potentiation (LTP) is characterized by a long lasting increase in the efficacy of neurotransmission which may consist of two phases. First an induction phase, with an absolute requirement for post-synaptic activation. Second, a maintenance phase, possibly involving pre-synaptic mechanisms. An essential function for calcium ions in the induction of LTP has been established and a particular emphasis has been placed on the role of N-methyl-D-aspartate (NMDA) receptor activation in gating a postsynaptic influx of calcium. We now report that pharmacological blockade of intraneuronal calcium release with 20 microM dantrolene-sodium (dantrium) completely blocks the induction of LTP in the CA1 region of the rat hippocampal slice. This drug inhibits calcium release from the sarcoplasmic reticulum and also diminishes the rise in intraneuronal calcium ion concentrations elicited by NMDA receptor activation in cultured CA1 pyramidal cells. Dantrolene does not block NMDA gated membrane currents or voltage activated Ca2+ currents in these cells. We suggest that release of intraneuronal calcium, rather than calcium influx may be the critical post-synaptic feature underlying LTP induction. We do not however exclude a pre-synaptic involvement in the specificity and/or maintenance of long-term potentiation.

Mitochondrial Sensitivity and Altered Calcium Handling Underlie Enhanced NMDA-Induced Apoptosis in YAC128 Model of Huntington's Disease

Expansion of a CAG repeat in the Huntingtons disease (HD) gene results in progressive neuronal loss, particularly of striatal medium-sized spiny neurons (MSNs). Studies in human HD autopsy brain tissue, as well as cellular and animal models of HD, suggest that increased activity of NMDA-type glutamate receptors and altered mitochondrial function contribute to selective neuronal degeneration. In this regard, the YAC128 mouse model, expressing full-length human huntingtin with 128 glutamine repeats, has been the focus of much interest. Although NMDA-induced apoptosis is enhanced in YAC128 MSNs, here we report that the initial steps in the death signaling pathway, including NMDA receptor (NMDAR) current and cytosolic Ca2+ loading, are similar to those observed in wild-type MSNs. In contrast, we found that the NMDAR-mediated Ca2+ load triggered a strikingly enhanced loss of mitochondrial membrane potential in YAC128 MSNs, suggesting that NMDAR signaling via the mitochondrial apoptotic pathway is altered. This effect was accompanied by impaired cytosolic Ca2+ clearance after removal of NMDA, a difference that was not apparent after high potassium-evoked depolarization-mediated Ca2+ entry. Inhibition of the mitochondrial permeability transition (mPT) reduced peak cytosolic Ca2+ and mitochondrial depolarization evoked by NMDA in YAC128 MSNs but not wild-type MSNs. Hence, in contrast to YAC models with moderate CAG expansions, the enhanced NMDA-induced apoptosis in YAC128 MSNs is predominantly determined by augmented mitochondrial sensitivity to Ca2+-induced activation of the mPT. These results suggest that the CAG repeat length influences the mechanism by which mHtt enhances NMDAR-mediated excitotoxicity.