Search

Studies over the past decade have demonstrated that lactate is produced aerobically during brain activation and it has been suggested to be an obligatory aerobic energy substrate postischemia. It has been also hypothesized, based on in vitro studies, that lactate, produced by glia in large amounts during activation and/or ischemia/hypoxia, is transported via specific glial and neuronal monocarboxylate transporters into neurons for aerobic utilization. To test the role of lactate as an aerobic energy substrate postischemia in vivo, we employed the cardiac-arrest-induced transient global cerebral ischemia (TGI) rat model and the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN). Once 4-CIN was establish to cross the blood--brain barrier, rats were treated with the inhibitor 60 min prior to a 5-min TGI. These rats exhibited a significantly greater degree of delayed neuronal damage in the hippocampus than control, untreated rats, as measured 7 days post-TGI. We concluded that intra-ischemically-accumulated lactate is utilized aerobically as the main energy substrate immediately postischemia. Blockade of lactate transport into neurons prevents its utilization and, consequently, exacerbates delayed ischemic neuronal damage.

Experiments are described in which a rat hippocampal slice preparation was used along with the metabolic glial inhibitor, fluorocitrate (FC), to investigate the role of glial-made lactate and its shuttling to neurons in posthypoxia recovery of synaptic function. After testing two less effective concentrations of FC, only 10.1 +/- 6.5% of slices treated with 100 microM of the metabolic toxin recovered synaptic function at the end of 10-min hypoxia and 30-min reoxygenation. In contrast, 79.6 +/- 7.4% of control, untreated slices recovered synaptic function after 10-min hypoxia and 30-min reoxygenation. The low rate of recovery of synaptic function posthypoxia in FC-treated slices occurred despite the abundance of glucose present in the medium before, during, and after hypoxia. The amount of lactate produced by FC-treated slices during the hypoxic period was only 62% of that produced by control, untreated slices. Supplementing FC-treated slices with exogenous lactate significantly increased the posthypoxia recovery rate of synaptic function. These results strongly support our previous findings concerning the mandatory role of lactate as an aerobic energy substrate for the recovery of synaptic function posthypoxia and clearly show that the bulk of the lactate needed for this recovery originates in glial cells.

Lactate has been considered for many years to be a useless, and frequently, harmful end-product of anaerobic glycolysis. In the present in vitro study, lactate-supplied rat hippocampal slices showed a significantly higher degree of recovery of synaptic function after a short hypoxic period than slices supplied with an equicaloric amount of glucose. More importantly, all slices in which anaerobic lactate production was enhanced by pre-hypoxia glucose overload exhibited functional recovery after a prolonged hypoxia. An 80% recovery of synaptic function was observed even when glucose utilization was blocked with 2-deoxy-D-glucose during the later part of the hypoxic period and during reoxygenation. In contrast, slices in which anaerobic lactate production was blocked during the initial stages of hypoxia did not recover their synaptic function upon reoxygenation despite the abundance of glucose and the removal of 2-deoxy-D-glucose. Thus, for brain tissue to show functional recovery after prolonged period of hypoxia, the aerobic utilization of lactate as an energy substrate is mandatory.

Changes in hippocampal calcium-binding protein (CaBP) were examined in rats given kindling stimuli via electrodes chronically implanted in the midline commissural pathway. CaBP levels decreased progressively and were significantly lower (16.6%) than controls after only 10 kindling trials. The maximum fall (33%) was achieved prior to the production of stage 5 motor seizures and additional kindling-induced seizures produced no further decline. Induction of motor seizures with pentylenetetrazol had no effect upon hippocampal CaBP levels. Diazepam treatment during the course of kindling significantly increased the number of stimulation trials required to produce stage 5 motor seizures but did not inhibit the fall in CaBP. Diazepam treatment of fully kindled rats was effective in blocking generalized motor seizures without causing any restoration of the depleted levels of CaBP. Diphenylhydantoin was neither effective during the course of kindling nor in modifying the effect of further stimulations in fully kindled rats. These data indicate that the highly specific decrease in hippocampal CaBP, previously demonstrated to be localized to dentate granule cells and their processes following kindling-induced epilepsy, does not result from the expression of full tonic-clonic (stage 5) motor seizures. The loss of CaBP may be a biochemical factor contributing either to the predisposition of neuronal tissue to seizure activity or to a protective attempt to overcome the deleterious effect of repeated high-frequency stimulation.

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.

Alterations in 45Ca uptake and retention as well as total intracellular calcium content were measured in hippocampal 'in vitro' slice preparations following the induction of long-term potentiation (LTP) in CA1 pyramidal neurones. The results indicate that tetanic stimulation of the Schaffer-commissural input produces a significant increase in the uptake and retention of calcium which parallels LTP of the CA1 population spike response.

The influence of extracellular fields on the transmembrane potential (TMP) of CA1 pyramidal neurons was investigated following both ortho- and antidromic stimulation in the in vitro hippocampal slice preparation. A short latency negative deflection on the intracellular potential coincided with the falling phase of the extracellular population spike. Subtraction of extracellular field potentials from ground referenced intracellular records revealed a sharp depolarizing wave of the TMP superimposed upon the underlying synaptic potential. This graded depolarization was capable of discharging CA1 cells and displayed a parallel shift in latency and amplitude with the extracellular population spike. A similar depolarizing wave was associated with the antidromically evoked population spike which persisted following blockade of synaptic activity. Finally, multiple population spike activity similar to that observed during epileptiform discharge was associated with repetitive depolarizing waves of the TMP. These data suggest that extracellular field potentials can ephaptically discharge CA1 neurons and may play a role in recruitment and synchronization of neuronal activity in the hippocampus.

The responses of dentate granule cells to medial septum (MS) and perforant path (PP) stimulation were examined in urethane anaesthetized rats. MS and PP stimulation evoked an orthodromic activation of granule cells which was correlated with the negative transient of the characteristic field potential elicited from each site. The effects of electrophoretic application of acetylcholine (ACh) and glutamate (Glu) were examined on granule cells identified in this manner. The excitatory action of ACh but not that of Glu was antagonized by atropine. Glutamate diethylester (GDEE) blocked the excitation produced by Glu but not ACh. The synaptically evoked excitation elicited by MS was blocked by atropine but unaltered by GDEE whereas the PP excitatory response was blocked by GDEE and unaltered by atropine. The results of this study indicate that two discrete excitatory systems are present in the dentate gyrus of the rat: a cholinergic system originating in the medial septum and a glutamate mediated system originating in the entorhinal cortex.

The levels and distribution of calcium binding protein (CaBP) were examined in the central nervous system of rats exhibiting kindling-induced epilepsy. Following commissural kindling, the concentration of CaBP in the hippocampal formation was significantly reduced but no change was observed in levels of total soluble protein or calmodulin, another calcium-related protein. Histochemical examination of kindled animals revealed a localized depletion of CaBP-like immunoreactivity restricted to the dentate granule cell-mossy fiber system of the hippocampal formation. These data demonstrate a correlation between the loss of CaBP from dentate granule cells and kindling-induced epilepsy, the underlying mechanism of which may involve an impairment of granule cells to regulate their intracellular calcium environment when challenged with high frequency electrical stimulation.

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.

The extracellular responses of neurones in the neostriatum following single pulse stimulation of the substantia nigra were investigated in urethane anaesthetized rats. Low intensity stimulation (less than 10 V) evoked single large amplitude spikes while higher intensities (10-20 V) elicit a high frequency burst of small amplitude spikes or waves. When spontaneous or glutamate-induced large spikes are recorded, nigral stimulation causes their inhibition coincidentally with the development of a burst. If the burst is prevented, the inhibitory response disappears. Both the nigral evoked inhibition and burst response are unaffected by iontophoretically or systemically administered antonists of dopamine or by chemical lesions of the dopamine-containing nigral neurones. The monosynaptic activation of large amplitude striatal neurones, which could also be identified antidromically by stimulation of the globus pallidus, was reversibly blocked by dopamine antagonists. It is concluded (a) that the burst responses are induced through the antidromic excitation of striatonigral axons within the striatum; (b) that the striatal neurones thus activated are inhibitory interneurones and (c) that the dopamine-containing neurones of the nigra make excitatory synaptic contact with a population of striatal output cells, some of which at least project to the globus pallidus.