Elevated glial brain-derived neurotrophic factor in Parkinson's diseased nigra.

We show the cellular distribution of immunoreactivity (IR) for brain-derived-neurotrophic-factor (BDNF), neurotrophin-3 (NT-3) and tyrosine kinase receptors TRKB and TRKC in idiopathic Parkinson's disease (IPD) and controls at post-mortem. In both groups, nigral neurons, astrocytes, ramified and amoeboid microglia expressed all antigens. Caudate-putamen neurons expressed all antigens except BDNF with similar distribution between groups. In IPD nigra, increased numbers of BDNF-IR and, less frequently, NT-3-IR ramified glia surrounded fragmented neurons, accompanied by BDNF-IR in surrounding neuropil. Amoeboid microglia were abundant only in IPD nigral scars. In IPD, glia might up-regulate neurotrophins in response to signals released from failing nigral neurons.

Inflammatory regulators in Parkinson's disease: iNOS, lipocortin-1, and cyclooxygenases-1 and -2.

Degeneration of dopaminergic neurons and focal gliosis are pathological hallmarks of Parkinson's disease and although the brain is described as immune-privileged focal immune reactions surround failing nigral neurons. We examined the cellular distribution of pro- and anti-inflammatory molecules in human parkinsonian and neurologically normal substantia nigra and caudate-putamen postmortem. An up-regulation of nitric oxide synthase- and cyclo-oxygenase-1- and -2-containing amoeboid microglia was found in parkinsonian but not control nigra. Astroglia contained low levels of these molecules in both groups. Lipocortin-1-immunoreactive amoeboid microglia were present within the astrocytic envelope of neurons adjacent to or within glial scars in parkinsonian nigra only. Lipocortin-1 is known to have neuroprotective and anti-inflammatory properties. Up-regulation of nitric oxide synthase is generally associated with neurodestruction whereas prostaglandin synthesis may be either neurodestructive or protective. The balance of these molecules is likely to be decisive in determining neuronal survival or demise.

Functional and molecular properties of human astrocytes in acute hippocampal slices obtained from patients with temporal lobe epilepsy.

PURPOSE: The specific role of glial cells in epilepsy is still elusive. In this study, functional properties of astrocytes were investigated in acute hippocampal brain slices obtained from surgical specimens of patients with drug-resistant temporal lobe epilepsy (TLE). METHODS: The patch-clamp technique together with a single-cell reverse transcription-polymerase chain reaction approach were used to combine functional and molecular analysis in the same individual cell in situ. RESULTS: In patients with Ammon's horn sclerosis, the glial current patterns resembled properties of immature astrocytes in rodent hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin-sensitive Na+ currents. Hyperpolarizing voltages elicited inward rectifier K+ currents. Comparative recordings were made in astrocytes from patients with lesion-associated TLE that lacked significant histopathological hippocampal alterations. The inward rectifier K+ current density was significantly smaller in astrocytes from the sclerotic group compared with lesion-associated TLE patients. CONCLUSIONS: During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears that astrocytes in human sclerotic tissue reexpress an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release and may thus contribute to seizure generation and spread in this particular condition of human TLE.

Glial heterogeneity in expression of the inwardly rectifying K(+) channel, Kir4.1, in adult rat CNS.

Previous electrophysiological evidence has indicated that astrocytes and oligodendrocytes express inwardly rectifying K(+) channels both in vitro and in vivo. Here, for the first time, we have undertaken light microscopic immunohistochemical studies demonstrating the location of one such channel, Kir4.1, in both cell types in regions of the rat CNS. Some astrocytes such as those in the deep cerebellar nuclei, Bergmann glia, retinal Müller cells, and a subset in hippocampus express Kir4.1 immunoreactivity, but not others including those in white matter. Oligodendrocytes also express this protein, strongly in perikarya and to a lesser extent in their processes. Expression of Kir4.1 in astrocytes and oligodendrocytes would enable these cells to clear extracellular K(+) through this channel, whereas nonexpressors might use other mechanisms.

De novo expression of lipocortin-1 in reactive microglia and astrocytes in kainic acid lesioned rat cerebellum.

An understanding of the role of reactive glia in the neurodegenerative/regenerative process requires a knowledge of the molecules synthesised by these cells following trauma. We investigated the cellular localisation of lipocortin-1 (LC-1), a putative neuroprotective agent, in cryostat sections of normal and kainic acid lesioned rat cerebellum. In the normal cerebellum lipocortin-1 immunoreactivity was detected in Purkinje cell bodies and molecular layer interneurons. Following kainic acid (1 microg) induced lesions, it was rapidly upregulated in activated microglia, from which it appeared to be secreted. At later time points it was detected in activated astrocytes. LC-1 protein levels were quantified by a sensitive and specific ELISA. Compared to control cerebellum, LC-1 levels were dramatically elevated following lesion, peaking at 3 days: 760% of basal (unlesioned) levels. In situ hybridisation studies revealed a marked upregulation of LC-1 mRNA at 1 and 3 days following the lesion, indicating the transient de novo synthesis of this protein, consistent with a localisation to microglia. In vitro studies, on cultured astrocytes and microglia, demonstrated high levels of intracellular LC-1 in both cell types. LC-1 was detected in microglial but not astrocytic, conditioned media, confirming the in vivo observations that activated microglia may secrete LC-1. Our data show that at early time points following excitotoxic lesion to the cerebellum, it is activated microglia that synthesise and possibly secrete this protein, suggesting an important role of this cell type in immunosuppression and neuroprotection following damage to the central nervous system.

Expression of COX-2 by normal and reactive astrocytes in the adult rat central nervous system.

We have used a previously characterized antiserum against cycloxygenase-2 (COX-2) together with cold methanol fixation to immunohistochemically locate the protein in astrocytes in rat brain. Although in cerebral cortex most enzyme was located in neuronal perikarya as previously described, a number of glial fibrillary acidic protein (GFAP)-positive astrocytes were also labeled. No COX-2-positive neurons were seen in the cerebellum, but here also a subset of GFAP+ astrocytes was present which contained the enzyme. The number of COX-2-positive astrocytes increased considerably after injection of the neurotoxin kainate into the cerebellum. These immunohistochemical data were supported by semiquantitative RT-PCR results, which were used to assess the levels of COX-2 mRNA relative to the housekeeping gene hypoxanthine phosphoribosyl transferase. PGE2 levels were measured in contralateral and lesioned cerebellum to correlate changes in COX-2 immunoreactivity and mRNA with physiological events. PGE2 levels increased by 230% in the lesioned cerebellar hemispheres in comparison to the contralateral ones. We discuss the possibility that the targets for astrocytic prostaglandins might include both autocrine effects and paracrine responses of neurons, lymphocytes and capillary endothelial cells.

Survival of genetically engineered, adult-derived rat astrocytes grafted into the 6-hydroxydopamine lesioned adult rat striatum.

Astrocytes are potentially useful as vehicles for gene transfer into the CNS. As endogenous CNS cells, they possess secretory mechanisms and can be grown in vitro. We have developed an animal model of this system using autologous astrocyte grafts in Fischer 344 rats. Cultured cells were infected with an adenoviral vector containing the reporter gene lacZ in vitro and then grafted into the striatum of adult Fischer 344 rats previously lesioned with 6-OHDA. Survival of the cells and activity of the beta-galactosidase protein were followed for up to 21 days after injection. The grafted cells were shown to survive throughout the experimental period although the expression of transgene was reduced with time. If long-term expression of therapeutically active substances can be achieved, grafts of adult-derived astrocytes genetically engineered using recombinant adenoviral vectors could be employed in the treatment of Parkinson's disease and other neurological disorders.

Astrocytes and microglia in the substantia nigra and caudate-putamen in Parkinson's disease.

There is evidence that astrocytes and microglia can release agents which might have a protective effect on damaged neurons. However the associations of glia with dopaminergic neurons in Parkinson's disease are not defined. Our studies showed that in post mortem parkinsonian nigra healthy neuronal somata were enveloped by astrocyte processes but these withdrew from fragmenting neurons and amoeboid microglia occupied the vacated peri-somal space. Thus, during the course of the disease both classes of glia are appropriately sited to release protective molecules onto failing neurons, but this may be inadequate for their long-term survival.

Cultured astrocytes express messenger RNA for multiple serotonin receptor subtypes, without functional coupling of 5-HT1 receptor subtypes to adenylyl cyclase.

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT2 family coupled to phospholipase C and of the 5-HT7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5B, 5-HT6 and 5-HT7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10-12 days. Messenger RNAs for 5-HT4 and 5-HT5A receptors were not detected. The functional expression of 5-HT1 receptor subtypes was investigated by measuring the ability of 5-HT1 receptor agonists: 8-OH-DPAT (5-HT1A receptors), RU24969 (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F receptors) or sumatriptan (5-HT1B, 5-HT1D, and 5-HT1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 microM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles.

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H]5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na+ dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised 'neuronal' serotonin transporter.

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.

Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes.

1. 5-Hydroxytryptamine (5-HT) elicited a dose-dependent stimulation of intracellular adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 +/- 0.08 (mean +/- s.e. mean). 2. In order to characterize the 5-HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5-carboxamidotryptamine (5-CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5-CT (pEC50 = 7.81 +/- 0.09) > 5-methoxytryptamine (5-MeOT) (pEC50 = 6.86 +/- 0.36) > 5-HT (pEC50 = 6.68 +/- 0.08). The following compounds, at concentrations up to 10 microM, did not affect basal cyclic AMP levels 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was methiothepin (pKi = 7.98 +/- 0.25) > mesulergine (pKi = 7.58 +/- 0.18) > ritanserin (pKi = 7.20 +/- 0.24) > clozapine (pKi = 7.03 +/- 0.19) > mianserin (pKi = 6.41 +/- 0.19). The following compounds, at concentrations up to 10 microM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5-HT7 receptor subtype-mediated effects. 3. The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 +/- 75% and 572 +/- 63% of basal levels for 5-CT and 5-HT, respectively) compared to brainstem (274 +/- 51% and 318 +/- 46%, respectively) and colliculus astrocytes (244 +/- 15% and 301 +/- 24%, respectively). No significant differences in pEC50 (for either 5-HT or 5-CT) values were observed. 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for the 5-HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5-HT6 receptor also amplified a cDNA fragment from the same samples. 5. From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5-HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally-dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5-HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5-HT6 and 5-HT7 receptor messenger RNA.

Ultrastructural localization of a voltage-gated K+ channel alpha subunit (KV 1.2) in the rat cerebellum.

A highly specific monoclonal antibody and pre-embedding immunocytochemistry were employed to examine the distribution of the K+ channel, alpha subunit K(V)1.2 in the rat cerebellum. At the light microscopic level, the heaviest immunoreactivity was seen in the basket cell pinceau at the base of Purkinje cells, with lighter staining of basket and Golgi cell bodies and a punctate pattern in the granule cell and molecular layers. Electron microscopy was performed to identify the ultrastructural location of K(V)1.2 alpha subunit in these labelled structures. This revealed that the labelling of the pinceau was confined to the preterminal axonal plexus, the area immediately around the Purkinje axon initial segment being relatively devoid of staining. Basket cell parent axons were not immunostained, but gave rise to heavily stained fine processes. Immunoreactivity was also seen in myelinated axons in the granule cell layer and in the medial cerebellar nucleus, the staining being most concentrated at the juxtaparanodal regions of the axons. An unusual pattern of staining was seen in some mossy fibre terminals, with staining restricted to fine protuberances of mossy fibre glomeruli. Structures contacted by these protuberances included adjoining glial processes. Immunostaining was absent from Purkinje cell bodies, dendrites, their axon initial segments and their terminals in the medial cerebellar nucleus. In this study, the alpha subunit K(V)1.2 was localized to a number of different cell types in the cerebellum. Each neuronal type displays a distinct subcellular distribution of the subunit.

Histochemistry of NADPH-diaphorase--a marker for neuronal nitric oxide synthase--in the carp retina.

The nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemical technique was used as a marker to assess the distribution of nitric oxide synthase activity in the carp retina. NADPH-diaphorase activity was found to be present in photoreceptors (rods and cones), horizontal cells, amacrine cells, bipolar cells, Müller cells and ganglion cells. Staining was most prominent in the photoreceptor ellipsoids but was not confined to any particular subtype. The density of the staining within the inner plexiform layer (IPL) was determined by image analysis. There was a broad peak of activity in each sublamina of the IPL, but sublamina b appeared to be relatively more heavily stained. The results taken together suggest that the nitric oxide signalling system could have a broader involvement in retinal function than previously thought. Furthermore, nitric oxide may have a novel mode of action in the retina whereby it could be effective on cells (photoreceptors) that also synthesize it.

Preprotachykinin-A and substance P receptor (NK1) gene expression in rat astrocytes in vitro.

The presence of mRNA transcripts coding for preprotachykinin-A and the substance P receptor in cultured astrocytes is demonstrated by a combination of reverse transcription/polymerase chain reaction (PCR) and Southern blotting. These findings showed that astrocytes in culture are capable of synthesing both the precursor of substance P (preprotachykinin-A) and the cognate receptor, substance P receptor (NK1). The simultaneous presence of both the ligand (substance P) and the receptor (NK1) may indicate an autocrine nature of astrocyte communication.

Serotonergic terminals express a growth associated protein (GAP-43) in the adult rat spinal cord.

Dual colour immunofluorescence has been used to compare the distribution of serotonin (5-hydroxytryptamine, 5-HT) and GAP-43 in the adult rat. GAP-43 immunostaining was observed in all spinal cord regions containing 5-HT immunoreactivity. 5-HT and GAP-43 double labelled fibres and varicosities were present and were most evident around motoneurones, in lamina X, and in the intermediolateral cell column. Single labelled GAP-43 fibres and varicosities were also observed and were the dominant population in the dorsal horn and in certain fibre tracts. We conclude that the 5-HT system is one of a small number of spinal cord systems that express high levels of GAP-43 in the adult.

Up-regulation of lipocortin-1 and its mRNA in reactive astrocytes in kainate-lesioned rat cerebellum.

We have used a combined molecular and immunocytochemical approach to examine the expression of lipocortin-1 (LC-1) in kainate-lesioned rat cerebellum. Using immunocytochemistry, Western and Northern blotting, we have shown upregulation of LC-1 mRNA and expression of LC-1 localised specifically to reactive astrocytes. These studies suggest that reactive astrocytes are a major synthetic compartment for the expression of LC-1. The well-reported immuno-suppressive effects of lipocortin(s), suggests that reactive astrocytes could serve to negatively modulate inflammatory reactions in the central nervous system.

Calcium-mobilizing and electrophysiological effects of bradykinin on cortical astrocyte subtypes in culture.

The possible consequences of activating bradykinin (Bk) receptors on identified astrocyte subtypes derived from rat cortex have been investigated in terms of: 1) mobilization of intracellular Ca2+; and 2) electrophysiological response. Bk induced a rapid, transient rise in intracellular Ca2+ in 63% of cortical type-1-like astrocytes and 44% of type-2 astrocytes tested. Experiments involving Ca(2+)-free conditions suggested that the release occurred largely from internal stores in both astrocyte subtypes. Bk receptor activation resulted in an inward current in approximately 10% of each astrocyte subtype tested using the whole-cell patch clamp technique. The use of perforated patch recording confirmed a similar population of cells responsive to Bk. The Bk-induced current was associated with a decrease in membrane conductance. The reversal potential was close to the K+ equilibrium potential, consistent with the closure of K+ channels. The current demonstrated similar EC50 values and Hill coefficients and also a marked degree of desensitization for both astrocyte subtypes. Recordings from type-1-like astrocytes indicated that the receptor mediating the electrophysiological response was of the B2 subtype. The data were consistent with the functional translation of Bk receptor occupation to physiological responses in distinct sub-populations of cortical type-1-like and type-2 astrocytes.

Prominent location of a K+ channel containing the alpha subunit Kv 1.2 in the basket cell nerve terminals of rat cerebellum.

A panel of monoclonal antibodies specific for a family of voltage-dependent, fast-activating K+ channels, raised against alpha-dendrotoxin acceptors purified from bovine brain, were used to probe the distribution of these important proteins in rat cerebellum. All the antibodies reacted with their antigens in the folial white matter, the granular cell layer and the basket cell nerve termini within the Purkinje cell layer. However, a very intense staining pattern was exhibited by only one monoclonal that reacts exclusively with Kv 1.2 alpha subunit, the predominant isoform present in alpha-dendrotoxin sensitive K+ channels. Double-labelling procedures with neuronal and glial markers were used to verify this discrete antibody staining of the basket cell terminals that synapse with the base of Purkinje cell bodies in a readily recognizable and characteristic fashion. This is the first direct demonstration, using a monoclonal antibody, of a presynaptic location for a voltage-activated K+ channel; its discrete distribution in the basket cell pinceau suggests that it could control release of the inhibitory transmitter GABA and, thereby, influence excitability of Purkinje cells in the cerebellum.