Martin Ingvar

Functional Activity of Substantia Nigra Grafts Reinnervating the Striatum: Neurotransmitter Metabolism and [14C]2-Deoxy-d-glucose Autoradiography

Abstract: Dopaminergic innervation of the caudate nucleus in adult rats can be partially restored by the grafting of embryonic substantia nigra into the overlying parietal cortex with concomitant compensation of certain behavioral abnormalities. In this study the function of such grafts was investigated neurochemically by quantification of transmitter metabolism and glucose utilization in the reinnervated target. Rats with unilateral 6‐hydroxydopamine lesions of the nigrostriatal bundle received a single graft to the dorsal caudateputamen and were screened for rotational behavior following 5 mg/kg methamphetamine. The grafts restored dopamine concentrations in the caudateputamen from initially less than 0.5% to an average of 13.6% of normal in rats with behavioral compensation. The ratio of 3,4‐dihydroxyphenylacetic acid to dopamine, which is a measure of the rate of transmitter turnover, were equivalent in transplanted and normal control rats. Moreover, measurements of DOPA accumulation for a 30‐min period after DOPA decarboxylase inhibition indicated similar fractional dopamine turnover rates in normal and transplantreinnervated tissues. Correlations between rotational behavior and dopamine concentrations showed that reinnervation to only 3% of normal was sufficient to counterbalance the motor asymmetry. Measurements of glucose utilization by [14C]deoxyglucose autoradiography indicated equivalent metabolic rates for the grafted tissue and the intact substantia nigra. 6‐Hydroxydopamine denervation of the caudate‐putamen had no significant effect on neuronal metabolism in that region, nor did subsequent reinnervation from a graft. Grafts, however, were associated with a 16% reduction of glucose uptake in the ipsilateral globus pallidus, indicating a significant transsynaptic influence of the nigral transplants on neuronal metabolism in the host brain. Overall the results indicate that behaviorally functional neuronal grafts spontaneously metabolize dopamine and utilize glucose at rates characteristic of the intact nigrostriatal system. This provides further evidence that ectopic intracortical nigral trans‐plants can reinstate dopaminergic neurotransmission in regions of the host brain initially denervated by the 6‐hydroxydopamine lesion.

Extra- and Intracellular pH in the Brain During Seizures and in the Recovery Period Following the Arrest of Seizure Activity

The objective of the study was to estimate changes in extracellular pH (pHe) and intracellular pH (pHi) during seizures and in the recovery period following the arrest of seizure activity. Seizures of 5- and 20-min duration were induced in rats by fluorothyl added to the insufflated gas mixture, and recovery for 5, 15, and 45 min was instituted by withdrawal of the fluorothyl supply following 20 min of continuous seizures. Changes in pHe were measured by double-barreled, liquid ion-exchange pH microelectrodes, and in pHi by the CO2 method, following estimation of tissue PCO2 and extracellular fluid (ECF) volume. The animals were either normoxic or rendered moderately hypoxic (arterial Po2 40–50 mm Hg). Upon induction of seizures in normoxic animals, pHe decreased by a mean of 0.36 unit, the values being identical at 5 and 20 min. In moderate hypoxia, seizures sustained for 20 min were accompanied by a further fall in pHe (mean decrease 0.51 unit). The changes in pHe seemed mainly to reflect the nonionic diffusion of lactic acid from cells to the ECF (tissue lactate levels ∼ 10 and 15 μmol g−1 during seizures in normoxic and hypoxic animals, respectively). However, the gradual fall in pHe attributable to lactic acid production was preceded by rapid acidification, sometimes exceeding the steady-state values subsequently attained. This acidification was interpreted to reflect spreading depression and fast transcellular Na+/H+ exchange. Following cessation of seizure discharge, pHe normalized at a surprisingly slow rate, with some acidosis persisting even after 45 min. The difference between cerebrovenous and arterial Pco2 was reduced during seizures and increased in the recovery period, probably reflecting alterations in the blood flow/metabolic rate coupling. Impedance changes were slight, indicating only minor changes in ECF volume. Changes in pHi after 5 min of seizures ranged from 0.20 (normoxic animals) to 0.32 (hypoxic animals) unit, the pHi values after 20 min being 0.07–0.08 unit higher. The results suggest the regulation of pHi during ongoing seizures. Upon arrest of seizure activity, pHi rapidly increased to normal and subsequently to supranormal values. Postepileptic intracellular alkalosis occurred at a time when pHe was still reduced and in spite of the fact that tissue lactate values had not normalized. It is concluded that the rapid normalization of pHi and overt alkalosis were caused by the simultaneously occurring oxidation of lactate, with the removal of a stoichiometrical amount of H+, and the extrusion of H+ from cells, possibly via a Na+/H+ exchanger, the latter probably delaying normalization of pHe.

Local blood flow and glucose consumption in the rat brain during sustained bicuculline‐induced seizures

The present study addresses the problem of whether brain structures which have been shown to develop neuronal cell damage in recurrent or prolonged epileptic seizures have higher metabolic rates and/or less pronounced increases in blood flow rates than others during sustained seizures. To that end, local cerebral blood flow (CBF) and glucose utilization (CMRgl) were measured autoradiographically in ventilated rats, in which seizures of 20, 60, or 120 min duration were induced by i.v. bicuculline.

The influence of bicuculline-induced seizures on free fatty acid concentrations in cerebral cortex, hippocampus, and cerebellum.

Abstract: Using ventilated rats maintained on N2O‐O2 (70:30, vol/vol) we induced continuous seizures with i.v. bicuculline and analysed free fatty acids (FFA) in cerebral cortex, hippocampus, and cerebellum after seizure durations of 1–120 min. In the cerebral cortex, peak FFA concentrations were observed after 5 min, with a threefold increase in total FFA content. The values then remained unchanged for the next 15‐20 min, but decreased thereafter. At 60 and 120 min, total FFA contents were only moderately increased above control. In the initial period, arachidonic acid increased about 10‐fold and stearic acid 2‐ to 3‐fold, with little change in palmitic acid and linoleic acid concentrations. At all times, the docosahexenoic acid concentration was markedly increased. Following its massive accumulation at 1 min, arachidonic acid gradually decreased in concentration. Pretreatment of animals with indomethacin did not alter this behaviour. After 20 and 120 min of seizure activity, changes in total and individual FFA concentrations in the hippocampus were similar to those observed in the cerebral cortex. The cerebellum behaved differently. Thus, at 20 min the only significant change was a 5‐ to 10‐fold increase in arachidonic acid concentration and, after 120 min, total and individual FFA concentrations were similar to control values. Furthermore, since the control values for arachidonic acid were much lower in the cerebellum, the 20‐min values were only about 20% of those observed in the cerebral cortex and the hippocampus.

Cerebral Blood Flow and Metabolic Rate during Seizures.: Relationship to Epileptic Brain Damage

UNLABELLED After long periods of status epilepticus, selective neuronal necrosis is incurred in the neocortex (layer III-IV), in the hippocampus (CA1 and CA4), and in the thalamus (VPL-VPM). In these areas the cerebral metabolic rate for glucose is increased to between 200-300% of control, indicating a correlation between neuronal damage and enhanced neuronal activity. Measurements of local cerebral blood flow indicate that the damage is not due to insufficient supply of oxygen. In most rats with status epilepticus lasting longer than 30 minutes, an infarction develops in the substantia nigra pars reticulata. In this region the metabolic rate is first increased but later during the seizure activity falls to very low values indicating cell necrosis. CONCLUSION prolonged neuronal hyperactivity with a concomitant increase in the metabolic rate for glucose is a prerequisite for the development of neuronal damage. However, the necrosis of the SNPR demonstrates that other factors determine the vulnerability of neurons to hyperexcitation, e.g., the type of agonist acting on the neuron.

Metabolic alterations underlying the development of hypermetabolic necrosis in the substantia nigra in status epilepticus

The substantia nigra pars reticulata (SNPR) has previously been shown to undergo tissue necrosis following status epilepticus induced by flurothyl in the rat. Even if the rat is ventilated, the SNPR develops necrosis if the epileptic period lasts more than 30 min. Rat brains were frozen in situ after 20 and 60 min of seizure activity and after 60 min of seizure activity followed by 60 min recovery. Labile energy metabolites were then analyzed in the SNPR and in the periaqueductal grey matter (PAG, control region). In the PAG, the metabolite changes during status epilepticus were similar to those reported for cerebral cortex and hippocampus. Measurements showed an unchanged ATP content and energy charge (97% and 98% of control, respectively) and an accumulation of lactate to 9.2 ± 0.6 μmol/g in the 60-min group. In the PAG, all metabolites measured had returned to control values after 60 min of recovery. In the SNPR, the perturbation of the energy metabolites was much more pronounced during status epilepticus. The concentration of ATP decreased to 75 ± 3%, the energy charge to 91% ± 12% and the adenylate pool to 86.7 ± 5.7% of control. Lactate accumulated to concentrations of 16.1 ± 1.8 μmol/g and 24.9 ± 2.3 μmol/g in the 20-min and 60-min groups, respectively. The concentration of lactate was still increased above control after 60 min recovery, whereas the concentration of ATP and the energy charge were lower than control. The findings demonstrate that sustained and intense neuronal activation can cause metabolic disturbance and thereby lead to necrosis. The very marked accumulation of lactic acid, likely due to mitochondrial failure, yields a very low intracellular pH, possibly explaining why a tissue necrosis, rather than selective neuronal necrosis, develops in the SNPR during status epilepticus.

Influence of Nitrous Oxide on Local Cerebral Blood Flow in Awake, Minimally Restrained Rats

In order to evaluate the effect of 70–80% N2O on local cerebral blood flow (l-CBF) in the rat brain, we developed a procedure for measuring CBF by an autoradiographic [14C]iodoantipyrine technique in awake, minimally restrained animals. Results on l-CBF, as measured in 22 different structures, showed little variability between animals. In the majority of structures analyzed, 70–80% N2O failed to alter l-CBF. These included all cerebral cortical and most subcortical structures. However, nitrous oxide reduced CBF in the the inferior colliculus and the superior olive, in two of the limbic structures analyzed, and in the hypothalamus. In no structure, except the striatum (p < 0.05), was a significant increase in l-CBF obtained in N2O-breathing animals. However, the results suggest that CBF may have been increased in the auditory cortex. Immobilization was found to reduce l-CBF in the cerebellum, inferior colliculus, superior olive, hippocampus, and septal nuclei. The results also suggest that the procedure somewhat increased CBF in frontal and parietal cortex. When the results obtained in awake, air-breathing animals were compared with those obtained in immobilized animals ventilated on N2O, there was no significant increase in any of the structures analyzed, although there were suggested increases in all cortical areas except the visual cortex. However, the data showed that ventilation with 70–80% N2O significantly decreased CBF in several structures (inferior colliculus, superior olive, hippocampus, amygdala, septal nuceli, and hypothalamus). In some of these, the effects of 70–80% N2O and of immobilization were obviously additive.

Regional Differences in Vascular Autoregulation in the Rat Brain in Severe Insulin-Induced Hypoglycemia

The present experiments were undertaken to determine if loss of vascular autoregulation during severe hypoglycemia shows regional differences that could help to explain the localization of hypoglycemic cell damage. Artificially ventilated rats (70% N2O) were subjected to a 30-min insulin-induced hypoglycemic coma (with cessation of EEG activity), with mean arterial blood pressure being maintained at 140, 120, 100, and 80 mm Hg. After 30 min of hypoglycemia, local cerebral blood flow (CBF) in 25 brain structures was measured autoradiographically with a [14C]iodoantipyrine technique. Since local CBF values did not differ between the 120 and the 100 mm Hg groups, the animals of these groups were pooled (110 mm Hg group). The results showed that at a blood pressure of 140 mm Hg, CBF was increased in 22 of 25 structures analysed, the maximal values approximating 300% of control. At 110 mm Hg, cerebral cortical structures had CBF values that were either decreased, normal, or slightly increased; however, many subcortical structures (and cerebellum) showed markedly increased flow rates. Although a lowering of blood pressure to 80 mm Hg usually further reduced flow rates, some of these latter structures also had well-maintained CBF values at that pressure. Thus, there were large interstructural variations of local CBF at any of the pressures examined. Analysis of the pressure–flow relationship showed loss of autoregulation in some structures, whereas others had remarkably well-preserved CBF values at low pressures. The results indicate that during severe hypoglycemia, even relatively moderate arterial hypotension may add a circulatory insult to the primary one, and they strongly suggest that any such insult affects some brain structures more than others.

Functional reactivation of the deafferented hippocampus by embryonic septal grafts as assessed by measurements of local glucose utilization

SummaryTransection of the septo-hippocampal connections through fimbria-fornix damage in the rat results in profound hippocampal cholinergic deafferentation, and, when applied bilaterally, leads to severe and long-lasting impairments in learning and memory. Previous studies have shown that intrahippocampal septal grafts can reestablish a new cholinergic innervation in the inititally denervated hippocampal formation and at least partly compensate for the lesion-induced learning impairments in fimbria-fornix lesioned rats. The purpose of the present study was to determine the magnitude of lesion-induced alterations in cerebral function as reflected in local glucose use measured by (14C)-2-deoxyglucose (2-DG) autoradiography, and the degree to which this index of functional activity could be normalized following reinnervation from transplants of fetal cerebral tissue from the primordial septal region. Six months after unilateral fimbriafornix transection the rate of glucose utilization was reduced markedly throughout the ipsilateral hippocampus when compared to the intact contralateral side, while in the neocortex only the cingulate cortex showed long-lasting reductions in glucose use. Rats that received a transplant of fetal septal-diagonal band tissue at the time of fimbria-fornix transection, and were sacrificed 6 months later, displayed significantly greater glucose utilization in the ipsilateral hippocampus and cingulate cortex than was measured in these areas in rats with lesion alone. The recovery in glucose use was paralleled by a significant increase in acetylcholinesterase (AChE) staining in several areas of the ipsilateral hippocampal formation and cingulate cortex. This index of graft-induced cholinergic reinnervation was, moreover, significantly correlated with the rate of glucose use. Thus, in the fimbria-fornix transected animals the magnitude of glucose depression correlated with the extent of reduction in AChE staining, and in the grafted animals the degree of normalization of glucose use was correlated with the graft-induced increase in AChE-staining density. These results thus indicate that the 2-DG autoradiographic technique can provide a unique opportunity to map both altered functional activity in localized areas of the brain following specific lesions and the extent to which transplant-derived reinnervation of the host may induce a return to normal functional levels in the target site.

Cellular and Molecular Events Underlying Epileptic Brain Damage

It has been known for more than 150 years that many patients with epilepsy can have unequivocal brain damage, usually localized to the hippocampus (for literature, see Reference 1). Such findings led to several important questions. The first was whether the brain damage was secondary to the seizures, or their cause. Since it subsequently emerged that repeated seizures and/or status epilepticus can in fact cause brain damage, the next important question arose: Is the damage due to the seizure discharge per se, or to systemic complications which encroach upon the oxygenation of brain cells? A related and equally pertinent question concerns the minimal period of seizure activity that carries the risk of inducing irreversible structural and functional lesions. We must ask, Can short electroconvulsive seizures, when administered singly or repetitively, cause such lesions? Many experimental protocols have been designed to provide appropriate answers to these questions (see Reference 2). Such experiments, as well as those designed to unravel the pathophysiology of ischemic or hypoglycemic brain damage, have yielded much useful information. As a result, we can now define in some detail pathophysiological conditions under which damage is incurred, and speculate about the mechanisms involved. It is the objective of this paper to discuss the neurochemical pathology of such damage.