Robert F. Ackermann

Increased seizure susceptibility of the immature brain

The ability of the CNS to generate seizures as a function of age was investigated utilizing the kindling model of epilepsy. Hourly electrical, low intensity stimulations of the amygdala induced kindling in adult rats, but stimulations delivered at 15 min intervals failed to or markedly retarded the development of kindled convulsions. In contrast, both types of stimulation induced consistent prolongation of the afterdischarges and repeated generalized seizures in suckling rat pups. The rate of development of the kindled convulsions in the pups was similar, irrespective of whether the stimulations were delivered at 15 or 60 min intervals, indicating that short (less than 15 min) seizure refractory periods exist in the immature brain. The data suggest that seizure susceptibility changes with age and is greater early in life.

Long term increase of glutamate decarboxylase mRNA in a rat model of temporal lobe epilepsy

Behavioral changes following injury, neural degeneration, and aging partly reflect the synaptic plasticity of the nervous system. Such long-term plastic changes are likely to depend on alterations in the production of proteins involved in synaptic structures and neurotransmission. We have studied the regulation of the mRNA encoding one such protein, glutamate decarboxylase (GAD), the rate limiting enzyme of GABA synthesis, after a unilateral lesion in the hippocampus that leads to increased seizure susceptibility. Quantitative in situ hybridization reveals a long-term increase in GAD mRNA in several bilateral structures, as well as in specific neurons in the ipsilateral dentate gyrus. Our data do not support the often stated hypothesis that seizure susceptibility depends on the malfunction of GABA neurons.

Glycolysis-Induced Discordance between Glucose Metabolic Rates Measured with Radiolabeled Fluorodeoxyglucose and Glucose

We have developed an autoradiographic method for estimating the oxidative and glycolytic components of local CMRglc (LCMRglc), using sequentially administered [18F]fIuorodeoxyglucose (FDG) and [14C]-6-glucose (GLC). FDG-6-phosphate accumulation is proportional to the rate of glucose phosphorylation, which occurs before the divergence of glycolytic (GMg) and oxidative (GMo) glucose metabolism and is therefore related to total cerebral glucose metabolism GMt: GMg + GMo = GMt. With oxidative metabolism, the 14C label of GLC is temporarily retained in Krebs cycle-related substrate pools. We hypothesize that with glycolytic metabolism, however, a significant fraction of the 14C label is lost from the brain via lactate production and efflux from the brain. Thus, cerebral GLC metabolite concentration may be more closely related to GMo than to GMt If true, the glycolytic metabolic rate will be related to the difference between FDG- and GLC-derived LCMRglc. Thus far, we have studied normal awake rats, rats with limbic activation induced by kainic acid (KA), and rats visually stimulated with 16-Hz flashes. In KA-treated rats, significant discordance between FDG and GLC accumulation, which we attribute to glycolysis, occurred only in activated limbic structures. In visually stimulated rats, significant discordance occurred only in the optic tectum.

In vivo [3H]spiperone binding: Evidence for accumulation in corpus striatum by agonist-mediated receptor internalization

The processes of receptor internalization and recycling have been well-documented for receptors for hormones, growth factors, lysosomal enzymes, and cellular substrates. Evidence also exists that these processes also occur for β-adrenergic, muscarinic cholinergic, and δ-opiate receptors in frog erythrocytes or cultured nervous tissue. In this study, evidence is presented that agonist-mediated receptor internalization and recycling occurs at the dopamine receptor in rat corpus striatum. First, the in vivo binding of the dopamine antagonist [3H]spiperone was increased by both electrical stimulation and pharmacologically induced increases of dopamine release. Conversely, depletion of dopamine with reserpine decreased in vivo [3H]spiperone binding, but the same reserpine treatment did not alter its in vitro binding. Second, the rate of dissociation of [3H]spiperone from microsomal membranes prepared from rat striatum following in vivo binding was fivefold slower than its dissociation following in vitro equilibrium binding. Mild detergent treatment, employed to disrupt endocytic vesicle membranes, increased the rate of dissociation of in vivo bound [3H]spiperone from microsomal membranes to values not significantly different from its in vitro bound dissociation rate. Third, treatment of rats with chloroquine, a drug that prevents receptor recycling but not internalization, prior to [3H]spiperone injection resulted in a selective increase of in vivo [3H]spiperone binding in the light microsome membranes. The existence of mechanisms that rapidly alter the number of neurotransmitter receptors at synapses provides dynamic regulation of receptors in response to varied acute stimulation states.

Pentylenetetrazol-induced kindling: early involvement of excitatory and inhibitory systems

Alterations in the brain of rats receiving a single non-convulsive administration pentylenetetrazol (PTZ), 30 mig/kg, i.p. (single PTZ group) were investigated and compared with those detected in fully PTZ kindled rats (chronic PTZ group). In vitro receptor autoradiography experiments showed that both single and chronic PTZ groups presented mu opioid and benzodiazepine (BDZ) receptor binding in specific brain areas. Using an antibody generated against the delta opioid receptor (DOR-1), it was found that DOR-1 like immunoreactivity was reduced in cortex and amygdala in mice following single and chronic PTZ administration. Microdialysis experiments revealed that the administration of PTZ 30 mg/kg, i.p. in freely moving rats without previous experience with the drug, induces a rise in glutamate release, detected in the first and second 10 min dialysates collected from amygdala (138% and 50%, respectively) and frontal cortex (70% and 45%, respectively) as well as aspartate in frontal cortex in the first and second PTZ-dialysates (143% and 80%, respectively). Subsequently, values returned to basal conditions. It may be speculated that decreased BDZ receptor binding results from enhanced release of GABA. On the other hand, the decrease of mu receptor binding and DOR-1 immunoreactivity observed after PTZ administration may be the result of enhanced levels of opioid peptides probably released over the kindling procedure. In conclusion, the present study indicates that PTZ-kindling is associated with an imbalance between excitatory and inhibitory systems which is apparent early in the epileptogenic process.

Functional [14C]2-deoxyglucose mapping of progressive states of status epilepticus induced by amygdala stimulation in rat

Electrical stimulation of rat amygdala induced self-sustained steady-state seizures (status epilepticus (SE] within 60 min. These SE states varied in behavioral severity from mere alteration of motility to frank clonic convulsions. Four distinct behavioral states were observed: immobility, exploration, mastication and clonus. These SE states were associated with [14C]2-deoxyglucose (2-DG) autoradiography anatomic patterns that were correspondingly more extensive and complex. Four distinct 2-DG activation patterns were observed: a restricted pattern involving several discrete limbic nuclei, including amygdala; more extensive patterns involving numerous limbic areas, first unilaterally, then bilaterally; finally the most extensive pattern involving widespread areas of forebrain. These data imply a systematic progression of seizure activity: originating in the amygdala, then spreading to some direct amygdala projection areas, and from there to a restricted network of interconnected ipsilateral limbic nuclei. This restricted network then recruits most of the remaining limbic structures, first ipsilaterally, then contralaterally. Finally, most of the basal ganglia, thalamus and neocortex are recruited.

Brain Mediation of Anolis Social Dominance Displays

Serotonin (5-HT) functions are associated with social dominance status in diverse species, but to date the brain regions wherein 5-HT exerts such effects are uncertain. Here, we indexed 5-HT turnover in male Anolis carolinensis as the ratio of 5-HT to its metabolite, 5-hydroxy-indol-acetic acid, and also as the accumulation of the in vivo tracer 14C-alpha-methyl-tryptophan (14C-AMT). After patching one eye, displaying dominant animals increased both measures of 5-HT turnover in the forebrain hemisphere receiving display-evocative visual stimuli, compared to control, contralateral brain, whereas both 5-HT turnover indices were decreased when animals displayed submissively. In contrast, various non-displaying controls showed forebrain symmetry on both measures. Drugs that stimulate 5-HT2C receptors in mammals, and have 5-HT2C-like binding in A. carolinensis, evoked some elements of dominant display behaviors in non-dominant anole males and also activated dorsolateral basal ganglia as seen in non-medicated dominants when they display [Baxter et al., 2001]. Thus, acute changes in forebrain 5-HT output from baseline equilibrium, acting at 5-HT2C-like receptors, might effect some elements of the dominant vs. submissive male anoles’ territorial displays. A mechanistic model of how this might occur is offered. Given similarities in 5-HT systems, forebrain functions, and territorial display routines, similar mechanisms might have similar functions in other amniotes, including primates.

Mapping of limbic seizure progressions utilizing the electrogenic status epilepticus model and the 14C-2-deoxyglucose method

We have previously described a model of limbic status epilepticus in which chronic prolonged seizure states of immobile, exploratory, minor convulsive or clonic convulsive behavior are induced by intracerebral electrical stimulation; these states appear to belong to the same behavioral progression as kindled seizures. We postulated that the underlying seizure substrates, as mapped by the 14C-2-deoxyglucose method, should reflect a corresponding anatomic progression of discharge spread. Status epilepticus was induced in rat by pulsed-train current delivered for up to 90 min to one of several subcortical areas. Autoradiographs revealed that most of the observed patterns of seizure-induced metabolic activation comprised a hierarchical sequence, such that progressively more extensive patterns subsumed anatomic territories activated in less extensive patterns, thus allowing inferences as to the progression of discharge spread. In this sequence, the basolateral amygdala ipsilateral to the induction electrode was among the first structures to be activated. In successively larger activation patterns a small unilateral network related to basolateral amygdala was involved; this evolved through a transitional state to a unilateral extensive limbic pattern; which in turn was succeeded by bilateral extensive limbic activation. This hierarchical sequence culminated in a neocortical activation pattern, in which most of the forebrain was involved in intense seizure-induced activation. Seizure behaviors increased in severity in correspondence with the underlying seizure-activated anatomic substrate. In contrast, patterns of seizure activation were observed which did not fit within the early stages of the above sequence, although analysis indicates that the later stages of spread may be shared. The study of these patterns and those reported in the literature indicates that although limbic seizure networks may be anatomically distinct at their origination, further expansion is characterized by overlap; upon assumption of extensive patterns of activation the number of nuclei participating is so vast that the identity of the limbic originator is lost and common convulsive manifestations occur.

Evaluation of [123I]isopropyliodoamphetamine as a tracer for local cerebral blood flow using direct autoradiographic comparison.

We investigated [123I]isopropyliodoamphetamine (IMP) for potential use in the autoradiographic determination of local cerebral blood flow (LCBF) in animals. The technique of direct autoradiographic comparison, derived from double radionuclide autoradiography, was used to compare the simultaneous uptakes of IMP and [14C]iodoantipyrine (IAP), a reference tracer, in awake and anesthetized rats. This new technique offers several advantages over the previously developed methods of comparing tracers, brain uptake index and first pass extraction ratio. These include the avoidance of disrupting normal cerebral blood–brain tracer exchange and the ability to compare uptakes at substructural levels, whereas the other methods are limited to larger areas. Mean values of LCBF obtained with IMP agreed closely with those using IAP, from 20 to 300 ml/100 g/min. Because IMP was found to have an extremely high effective brain:blood partition coefficient, approximately 25:1, a linear uptake tracer model could be used for IMP yielding more precise values than could IAP for LCBF values above 150. IMP was found to measure choroid plexus flows much more accurately than IAP, values being greater than 500 for IMP compared to approximately 200 for IAP. Because the mechanism of the extremely high partition coefficient of IMP is not yet defined, however, care must be used in measuring LCBF with IMP where the trapping mechanisms of normal vessels may be disrupted.

Increased susceptibility to hippocampal and amygdala kindling following intrahippocampal kainic acid

The effects of unilateral intrahippocampal injection of kainic acid, a potent neuroexcitant and neurotoxin, on subsequent susceptibility to kindling of the contralateral hippocampus or contralateral amygdala were investigated in albino rats. At the chosen doses (0.20 to 1.25 micrograms dissolved in physiologic saline), the kainic acid-induced lesion was confined to the injected hippocampus and in two cases the ipsilateral entorhinal cortex; never were there contralateral lesions. Approximately 2 to 6 weeks post-injection, each animal received daily afterdischarge-producing electrical stimulations until stage 5 kindled limbic seizures occurred. Kindling in pretreated animals was significantly accelerated compared with controls; the hippocampal kindling rate decreased from 13.2 stimulations to 3.7, the amygdala kindling rate from 7.8 stimulations to 3.0. Many treated animals had first-stimulation stage 5 seizures, compared with none for controls. Importantly, this facilitation of kindling was not reversed by suppression of the acute, induced seizures with the anticonvulsants, diazepam and phenobarbital, which have repeatedly been demonstrated to effectively suppress limbic kindling. Such results, considered together with findings from the literature, suggest that partial kindling does not occur during kainic acid-induced seizures, and that the observed susceptibility to kindling and other epileptogenic agents subsequent to kainic acid treatment may in fact be related to neurophysiologic and neurochemical consequences of kainic acid-induced lesions.