Stephen M. Sagar

Heat shock proteins as markers of neural injury.

Systemic or intracerebral injections of kainic acid induced immunoreactivity for the 72 kDa heat shock protein (HSP72) in individual neurons of the rat brain in patterns matching the known histopathology of the particular injury. HSP72 immunostaining was also induced in and around areas of infarction following experimental strokes. These results suggest that HSP72 immunocytochemistry may be used as a marker of cellular injury in the mammalian brain.

Sensory stimulation induces local cerebral glycogenolysis: demonstration by autoradiography.

Brain glycogen stores are localized primarily to glia and undergo continuous utilization and resynthesis. To study the function of glycogen under normal conditions in brain, we developed an autoradiographic method of demonstrating local-glycogen utilization in the awake rat. The method employs labeling of brain glycogen with 14C(3,4)glucose, in situ microwave fixation of brain metabolism, and anhydrous tissue preparation. With this technique, tactile stimulation of the rat face and vibrissae was found to accelerate the utilization of labeled glycogen in brain regions known to receive sensory input from face and vibrissae: the contralateral somatosensory cortex and the ipsilateral trigeminal, sensory and motor nuclei. These findings demonstrate a link between neuronal activity and local glycogen utilization in mammalian brain and suggest that, like other tissues, brain may respond to sudden increases in energy demand in part by rapid glycolytic metabolism of glycogen. As cerebral glycogen is restricted primarily to glia, these observations also support a close coupling of glial energy metabolism with neuronal activity.

Induction of 70-kDa heat shock protein and hsp70 mRNA following transient focal cerebral ischemia in the rat

Induction of the 70-kDa heat shock protein (HSP70) was demonstrated immunocytochemically in adult rats 4 h to 7 days following temporary middle cerebral artery (MCA) occlusions lasting 30, 60, or 90 min. Maximal HSP70 induction occurred ∼24 h following ischemia. Thirty minutes of ischemia induced HSP70 in neurons throughout the cortex in the MCA distribution, whereas 90 min of ischemia induced HSP70 in neurons in the penumbra. HSP70 protein was induced in endothelial cells in infarcted neocortex following 60–90 min of MCA occlusion, and HSP70 was induced in endothelial cells in infarcted regions of lateral striatum following 30–90 min of MCA occlusion. hsp70 mRNA was induced in the MCA distribution in cortex and to a lesser extent in striatum at 2 h to 3 days following 60 min of ischemia. It is proposed that brief ischemia induces hsp70 mRNA and HSP70 protein in the cells most vulnerable to ischemia—the neurons. HSP70 protein is not induced in most neurons and glia following 60–90 min of ischemia in areas destined to infarct, whereas it is induced in vascular endothelial cells.

Induction of c-Fos, junB, c-Jun, and hsp70 mRNA in Cortex, Thalamus, Basal Ganglia, and Hippocampus following Middle Cerebral Artery Occlusion

Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced c-fos, junB, and c-jun immediate early gene mRNAs and hsp70 heat shock gene mRNA in brain. In situ hybridization studies showed that c-fos and junB were induced throughout all of the cortex at 1 and 4 h following MCA occlusion. hsp70 was induced in the core and margins of the MCA ischemia. By 24 h, there was little expression of c-fos, junB, c-jun, and hsp70 in the core of the MCA infarct; there was modest induction of hsp70 at the margins of the infarct; and there was diffuse induction of c-fos, junB, and c-jun in all of the cortex outside the infarct. MCA occlusion also induced these genes in subcortical structures. c-fos, junB, and hsp70 were induced in ipsilateral medial striatum, most of thalamus including medial and lateral geniculate nuclei, substantia nigra, and hippocampus. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in gene expression can occur in regions remote from an infarction.

Light induces a Fos0like nuclear antigen in retinal neurons

Fos, the product of the proto-oncogene c-fos, is a nuclear phosphoprotein thought to participate in transcriptional regulation of target genes. To explore the synaptic induction of Fos and related proteins in vivo, Fos immunohistochemistry was performed in the rabbit retina. Dark-adapted retinas had virtually no Fos immunostaining. Retinas of dark-adapted rabbits that were exposed to 3 Hz diffuse flashing white light for 1 h and sacrificed 2 h later displayed nuclear Fos immunostaining in a minority of neurons. These included presumptive amacrine cells of the inner nuclear layer and either displaced amacrine cells or ganglion cells of the ganglion cells of the ganglion cell layer. Therefore, Fos or a related antigen is expressed in a subset of retinal neurons in response to light and is presumably involved in regulating gene expression of these cells to respond to alterations in synaptic activity.

NADPH diaphorase activity in the posterior pituitary: relation to neuronal function

Magnocellular neurons of the paraventricular and supraoptic nuclei, fibers of the medial basal hypothalamus, and the posterior pituitary gland all stain histochemically for NADPH diaphorase activity (NADPHd). Following 8 days of salt loading to stimulate the hypothalamo-neurohypophyseal system, NADPHd activity, as determined by a spectrophotometric assay, is markedly increased in the posterior pituitary gland but not in the hypothalamus of rats. Therefore, NADPHd activity in this system correlates with neuronal function and may provide a convenient method for the assessment of neuronal activity in selected neuronal populations.

The NMDA receptor mediates cortical induction of fos and fos-related antigens following cortical injury

Cortical cavity lesions and lateral ventricular injections of quinolinic acid, a NMDA receptor agonist, induce Fos and Fos-related antigens (FRAs) throughout ipsilateral adult rat brain cortex in similar patterns. c-fos mRNA, assessed using in situ hybridization, was induced by 1 h and disappeared between 3 and 8 h following cortical lesions. Fos proteins, detected using a specific monoclonal antibody, were induced by 1 h and disappeared by 4 h after cortical lesions. FRA proteins, detected using polyclonal antibodies, were induced between 1 and 4 h and persisted for at least 72 h following focal cortical injury. Intraventricular injections of CPP, a competitive NMDA receptor antagonist, completely blocked the induction of these nuclear proteins in cortex ipsilateral to the focal cortical lesions--except around the injury site itself. Intraventricular injections of quisqualate, a non-NMDA glutamate analogue, induced Fos in hippocampus but not in cortex. These data show that NMDA receptors mediate the induction of Fos and FRAs following cortical injury. It is proposed that local cortical injury releases excitatory amino acids that act at NMDA receptors to initiate spreading depression and that the resultant depolarization induces Fos in neurons throughout the cortex. Since Fos and FRAs are proteins that regulate the expression of target genes, they could mediate long-term biochemical adaptations in neurons following cortical injury.

NADPH diaphorase histochemistry in the rabbit retina

NADPH diaphorase activity has been shown by histochemical staining to co-localize with markers for selective neurotransmitter candidates in various regions of the rat brain. The rabbit retina was therefore examined to determine if the technique stains a selective population of retinal neurons as well. Whole retinas of adult, male, pigmented rabbits are incubated with a specific reaction mixture containing nitro blue tetrazolium as the electron acceptor. Dark blue reaction product is deposited in two populations of cell bodies near the inner border of the inner nuclear layer (INL). One cell type is larger and more darkly stained than the second. The larger cells have 2-4 tapering primary dendrites which branch sparsely in the inner plexiform layer (IPL) and which can be traced for up to 500 microns. The second cell type has smaller and more lightly stained somata. In retinal cross sections, a dense layer of varicose fibers is seen in the middle (sublamina 3) of the IPL; these fibers arise at least in part from the larger, darkly stained cell bodies. A less dense plexus of fibers is stained at the outer margin (sublamina 1) of the IPL, and occasional varicosities are seen in the inner sublaminas (4 and 5) of the IPL. NADPH diaphorase histochemistry, therefore, selectively stains at least two subtypes of amacrine cells in rabbit retina. Although a definite identification of the transmitter content of these cells cannot be made, diaphorase histochemistry provides, in the retina, a remarkably convenient method for achieving Golgi-like images of morphologically distinct neuronal populations.

NADPH-diaphorase (nitric oxide synthase)-reactive amacrine cells of rabbit retina : putative target cells and stimulation by light

In the mammalian retina there are two populations of nitric oxide synthase-containing amacrine cells that stain with the nicotinamide adenine dinucleotide phosphate-diaphorase reaction. To determine the response of these neurons to light, immunoreactivity to Fos proteins was used as a marker of synaptic activation. Fos immunoreactivity is absent in dark-adapted retinas, but 70% of large, Type I nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells and 5-10% of the smaller but more numerous Type II nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells contain Fos proteins after light stimulation. To localize putative cellular targets of nitric oxide in the retina, retinas were stained immunocytochemically for cyclic GMP after the local administration of the nitric oxide donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. Both compounds induce strong cyclic GMP immunoreactivity in ON cone bipolar cells. The data suggest that the light-induced inward current in ON cone bipolar cells is enhanced by a nitric oxide-cyclic GMP pathway and that the major source of nitric oxide is the nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells in the rabbit retina.

Phencyclidine induction of the hsp70 stress gene in injured pyramidal neurons is mediated via multiple receptors and voltage gated calcium channels

Non-competitive N-methyl-D-aspartate receptor antagonists, including phencyclidine, ketamine, and MK801, produce vacuoles and induce the hsp 70 stress gene in layer III pyramidal neurons of the rat cingulate cortex. This study shows that phencyclidine (50 mg/kg) induces hsp 70 messenger RNA and HSP70 stress protein primarily in pyramidal neurons in posterior cingulate and retrosplenial cortex, neocortex, insular cortex, piriform cortex, hippocampus, and in the basal nuclei of the amygdala. Several neurotransmitter receptor antagonists inhibited induction of HSP70 produced by phencyclidine (50 mg/kg): haloperidol (ED50 = 0.8 mg/kg), clozapine (ED50 = 1 mg/kg), valium (ED50 = 1 mg/kg), SCH 23390 (ED50 = 7 mg/kg) and muscimol (ED50 = 3 mg/kg). Baclofen had no effect. Nifedipine blocked the induction of HSP70 produced by phencyclidine in some regions (cingulate, neocortex, insular cortex) but only partially blocked HSP70 induction in other regions (piriform cortex, amygdala). These results suggest that phencyclidine injuries pyramidal neurons via dopamine D1, D2, D4, sigma and other receptors. Several factors appear to contribute to this unusual multi-receptor mediated injury. (1) Phencyclidine blocks N-methyl-D-aspartate receptors on GABAergic interneurons resulting in decreased inhibition of pyramidal neurons. This may help to explain why multiple excitatory receptors mediate the injury and why GABAA agonists decrease the injury produced by phencyclidine. (2) Phencyclidine blockade of an amine transporter helps explain why dopamine receptor antagonists ameliorate injury. (3) Phencyclidine depolarizes neurons and produces high, potentially damaging intracellular calcium levels probably by blocking K+ channels that may be linked to sigma receptors. Since nifedipine prevents injury in cingulate, insula, and neocortex, it appears that calcium entry through L-type voltage gated calcium channels plays a role in the pyramidal neuronal injury produced by phencyclidine in these regions. There are similarities between the cingulate neurons injured by phencyclidine and circuits recently hypothesized to explain receptor changes in cingulate gyrus of schizophrenic patients. The present and previous studies also provide approaches for decreasing the clinical side effects of N-methyl-D-aspartate receptor antagonists to facilitate their possible use in the treatment of ischemia and other disorders.