The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity.

We used purified cultures of cerebellar granule cells to investigate the possible protective role of stress proteins in an in vitro model of excitotoxicity. Initial experiments used one- and two-dimensional polyacrylamide gel electrophoresis to confirm the induction of typical stress protein size classes by heat shock, sodium arsenite, and the calcium ionophore A23187. Immunoblot analysis and immunocytochemistry verified the expression of the highly inducible 72 kd heat shock protein (HSP72). Granule cell cultures exposed to glutamate showed evidence of cellular injury that was prevented by the noncompetitive NMDA antagonist MK-801, yet glutamate did not induce a detectable stress protein response. Nonetheless, preinduction of heat shock proteins was associated with protection from toxic concentrations of glutamate. These results imply that the HSP72 expression observed in in vivo models of excitotoxicity may not be directly related to the effects of excitatory amino acids. However, the ability of stress protein induction to protect against injury from glutamate may offer a novel approach toward ameliorating damage from excitotoxins.

Up regulation of calbindin-D28K mRNA in the rat hippocampus following focal stimulation of the perforant path.

Calbindin-D28K is a constitutive Ca2(+)-binding protein expressed in hippocampal neurons that are resistant to various forms of excitotoxic injury. However, the local factors controlling calbindin-D28K expression within the central nervous system are unknown. We report that neuronal excitation via the perforant path leads to an increased expression of calbindin-D28K mRNA within dentate granule cells. This response is related specifically to stimulation that induces prolonged periods of bursting afterdischarges and precedes cellular injury. The up regulation of calbindin-D28K mRNA occurs during the type of neuronal activation associated with elevated cytosolic Ca2+ and suggests that the maintenance of Ca2+ homeostasis includes a system of feedback control at the level of gene expression.

Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures.

K+ channels are major determinants of membrane excitability. Differences in neuronal excitability within the nervous system may arise from differential expression of K+ channel genes, regulated spatially in a cell type-specific manner, or temporally in response to neuronal activity. We have compared the distribution of mRNAs of three K+ channel genes, Kv1.1, Kv1.2, and Kv4.2 in rat brain, and examined activity-dependent changes following treatment with the convulsant drug pentylenetetrazole. Both regional and cell type-specific differences of K+ channel gene expression were found. In addition, seizure activity caused a reduction of Kv1.2 and Kv4.2 mRNAs in the dentate granule cells of the hippocampus, raising the possibility that K+ channel gene regulation may play a role in long-term neuronal plasticity.

Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons.

GABAergic interneurons have major roles in hippocampal function and dysfunction. Here we provide evidence that, in mice, virtually all of these cells originate from progenitors in the basal telencephalon. Immature interneurons tangentially migrate from the basal telencephalon through the neocortex to take up their final positions in the hippocampus. Disrupting differentiation in the embryonic basal telencephalon (lateral and medial ganglionic eminences) through loss of Dlx1/2 homeobox function blocks the migration of virtually all GABAergic interneurons to the hippocampus. On the other hand, disrupting specification of the medial ganglionic eminence through loss of Nkx2.1 homeobox function depletes the hippocampus of a distinct subset of hippocampal interneurons. Loss of hippocampal interneurons does not appear to have major effects on the early development of hippocampal projection neurons nor on the pathfinding of afferrent tracts.

Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins.

Given the critical role of the prion protein (PrP) in the transmission and pathogenesis of experimental scrapie, we investigated the PrP gene and its protein products in three hamster species, Chinese (CHa), Armenian (AHa), and Syrian (SHa), each of which were found to have distinctive scrapie incubation times. Passaging studies demonstrated that the host species, and not the source of scrapie prions, determined the incubation time for each species, and histochemical studies of hamsters with clinical signs of scrapie revealed characteristic patterns of neuropathology. Northern (RNA) analysis showed the size of PrP mRNA from CHa, AHa, and SHa hamsters to be 2.5, 2.4, and 2.1 kilobases, respectively. Immunoblotting demonstrated that the PrP isoforms were of similar size (33 to 35 kilodaltons); however, the monoclonal antibody 13A5 raised against SHa PrP did not react with the CHa or AHa PrP molecules. Comparison of the three predicted amino acid sequences revealed that each is distinct. Furthermore, differences within the PrP open reading frame that uniquely distinguish the three hamster species are within a hydrophilic segment of 11 amino acids that includes polymorphisms linked to scrapie incubation times in inbred mice and an inherited prion disease of humans. Single polymorphisms in this region correlate with the presence or absence of amyloid plaques for a given hamster species or mouse inbred strain. Our findings demonstrate distinctive molecular, pathological, and clinical characteristics of scrapie in three related species and are consistent with the hypothesis that molecular properties of the host PrP play a pivotal role in determining the incubation time and neuropathological features of scrapie.

Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat.

Aberrant reorganization of dentate granule cell axons, the mossy fibers, occurs in human temporal lobe epilepsy and rodent epilepsy models. Whether this plasticity results from the remodeling of preexisting mossy fibers or instead reflects an abnormality of developing dentate granule cells is unknown. Because these neurons continue to be generated in the adult rodent and their production increases after seizures, mossy fibers that arise from either developing or mature granule cells are potential substrates for this network plasticity. Therefore, to determine whether seizure-induced, mossy fiber synaptic reorganization arises from either developing or mature granule cell populations, we used low-dose, whole-brain x-irradiation to eliminate proliferating dentate granule cell progenitors in adult rats. A single dose of 5 Gy irradiation blocked cell proliferation and eliminated putative progenitor cells in the dentate subgranular proliferative zone. Irradiation 1 d before pilocarpine-induced status epilepticus significantly attenuated dentate granule cell neurogenesis after seizures. Two irradiations, 1 d before and 4 d after status epilepticus, essentially abolished dentate granule cell neurogenesis but failed to prevent mossy fiber reorganization in the dentate molecular layer. These results indicate that dentate granule cell neurogenesis in the mature hippocampal formation is vulnerable to the effects of low-dose ionizing irradiation. Furthermore, the development of aberrant mossy fiber remodeling in the absence of neurogenesis suggests that mature dentate granule cells contribute substantially to seizure-induced network reorganization.

Hippocampal heterotopia lack functional Kv4.2 potassium channels in the methylazoxymethanol model of cortical malformations and epilepsy.

Human cortical malformations often result in severe forms of epilepsy. Although the morphological properties of cells within these malformations are well characterized, very little is known about the function of these cells. In rats, prenatal methylazoxymethanol (MAM) exposure produces distinct nodules of disorganized pyramidal-like neurons (e.g., nodular heterotopia) and loss of lamination in cortical and hippocampal structures. Hippocampal nodular heterotopias are prone to hyperexcitability and may contribute to the increased seizure susceptibility observed in these animals. Here we demonstrate that heterotopic pyramidal neurons in the hippocampus fail to express a potassium channel subunit corresponding to the fast, transient A-type current. In situ hybridization and immunohistochemical analysis revealed markedly reduced expression of Kv4.2 (A-type) channel subunits in heterotopic cell regions of the hippocampus of MAM-exposed rats. Patch-clamp recordings from visualized heterotopic neurons indicated a lack of fast, transient (I(A))-type potassium current and hyperexcitable firing. A-type currents were observed on normotopic pyramidal neurons in MAM-exposed rats and on interneurons, CA1 pyramidal neurons, and cortical layer V-VI pyramidal neurons in saline-treated control rats. Changes in A-current were not associated with an alteration in the function or expression of delayed, rectifier (Kv2.1) potassium channels on heterotopic cells. We conclude that heterotopic neurons lack functional A-type Kv4.2 potassium channels and that this abnormality could contribute to the increased excitability and decreased seizure thresholds associated with brain malformations in MAM-exposed rats.

Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS.

Wnt signaling regulates a wide range of developmental processes such as proliferation, cell migration, axon guidance, and cell fate determination. In this report, we studied the expression of secreted frizzled related protein-2 (SFRP-2), which codes for a putative Wnt inhibitor, in the developing nervous system. SFRP-2 is expressed in several discrete neuroepithelial domains, including the diencephalon, the insertion of the eminentia thalami into the caudal telencephalon, and the pallial-subpallial boundary (PSB). We also noted that Wnt-7b expression was similar to SFRP-2 expression. Because many of these structures are disrupted in Pax-6 mutant mice, we examined SFRP-2 and Wnt-7b expression in the forebrains of Pax-6 Sey/Sey mice. We found that Pax-6 mutants lack SFRP-2 expression in the PSB and diencephalon. Interestingly, Pax-6 mutants also lack Wnt-7b expression in the PSB, but Wnt-7b expression in the diencephalon is preserved. Furthermore, in the spinal cord of Pax-6 mutants, SFRP-2 and Wnt-7b expression was greatly reduced. Our results suggest that by virtue of its apposition to Wnt-7b expression, SFRP-2 may modulate its function, particularly at boundaries such as the PSB, and that changes in Wnt signaling contribute to the phenotype of Pax-6 mutants.

Wnt receptors and Wnt inhibitors are expressed in gradients in the developing telencephalon.

The caudomedial margin of the medial pallium, known as the cortical hem, expresses several Wnt genes that have been shown to be crucial for cortical development. We examined the expression of members of the Frizzled (mFz) family of Wnt receptors and the Secreted Frizzled Related Protein (SFRP) family of Wnt inhibitors during telencephalic development. We found that mFz-5 and mFz-8 are specifically expressed in the neocortical neuroepithelium and excluded from the hippocampal neuroepithelium in early telencephalic development, whereas mFz-9 and mFz-10 have expression domains confined to the medial pallium. In addition, SFRP-1 and SFRP-3 are expressed in opposing anterolateral to caudomedial gradients within the telencephalic ventricular zone throughout corticogenesis.

Heat shock protein hsp72 induction in cortical and striatal astrocytes and neurons following infarction.

Transient global and transient focal ischemia induced the 72 kDa heat shock protein (hsp72) in neurons in cortex, striatum, and other regions known to be injured during transient ischemia. A novel finding was the induction of hsp72 in islands (cylinders in three dimensions) of cells composed of astrocytes around the perimeter and neurons in the interior. Since histology showed pale staining in these regions, it is proposed that these islands represent areas of focal infarction in the distribution of small cortical and lenticulostriate arteries. Although the factors responsible for hsp72 induction during ischemia and infarction are unknown, these results suggest differences in mechanisms of hsp72 induction in astrocytes compared to neurons.

Regional induction of c-fos and heat shock protein-72 mRNA following fluid-percussion brain injury in the rat.

To evaluate the cellular response to traumatic brain injury, the expression of mRNA for c-fos and the 72-kDa heat shock protein (hsp72) was determined using in situ hybridization following lateral fluid-percussion injury (2.2-2.4 atm) in rat brain. At 2 h after injury, induction of c-fos mRNA was observed throughout the cortex ipsilateral to the site of injury, while increased expression of hsp72 mRNA was restricted to regions of the cortex surrounding the contusion area. An increase in c-fos mRNA, but not hsp72 mRNA, was observed bilaterally in the CA3 subfield of the hippocampus and the granule cells of the dentate gyrus and in the thalamus ipsilateral to the impact site. By 6 h, increased expression of c-fos mRNA was observed only in the corpus callosum on the impact side; hsp72 mRNA persisted in the deep cortical layers and upper layers of the subcortical white matter below the site of maximal injury. By 24 h, both c-fos and hsp72 mRNA had returned to control levels in all regions of the brain. These results demonstrate that lateral fluid-percussion brain injury triggers regionally and temporally specific expression of c-fos and hsp72 mRNA, which may be suggestive of differential neurochemical alterations in neurons and glia following experimental brain injury.

The clinical course of spasmodic torticollis.

Among 24 of 36 patients with idiopathic spasmodic torticollis referred to one of us over a 10-year period, who were followed up for more than 1 year, we defined three outcome groups. Three patients (13%) underwent complete or almost complete remission at a median of 3.0 years into the illness. Eight patients (33%) had partial remissions, which tended to occur somewhat later than the first group. The remaining 13 patients had no significant improvement in their disease; they were more likely to be older at the onset of the illness, develop constant rather than intermittent neck deviation, and have a "geste antagonistique." As a whole, the study population was exposed to a wide variety of therapeutic interventions that had little, if any, obvious clinical effect.

Clinical and EEG features of status epilepticus in comatose patients.

We retrospectively evaluated the clinical and EEG features of status epilepticus (SE) in 47 comatose adult patients in whom SE was suspected clinically or because the EEG revealed repetitive electrographic seizures or continuous spike-and-wave activity. Three groups of patients were identified. Group-1 patients (n = 33) had SE both clinically and on EEG. They usually had subtle, clonic movements restricted to the eyes, face, and upper extremities, and the EEG most commonly showed repetitive electrographic seizures or continuous spike-and-wave activity. Group-2 patients (n = 9) also had subtle motor manifestations of seizures, but the EEG was not that of SE, consisting of either irregular slowing with frequent spikes and sharp waves, an irregular mixed-frequency background with episodic accentuation, or diffuse slowing; one patient also had an intermittent burst-suppression pattern. The five patients in Group 3 lacked any clinical signs of seizures, but the EEG showed repetitive electrographic seizures or continuous spike-and-wave activity. There were no significant differences between groups in etiology of SE, response to therapy, or outcome, and there was no obvious relationship between the EEG findings and duration of SE. We conclude that recognition of SE in comatose patients may require both clinical and EEG evaluation since either approach by itself may fail to establish the diagnosis. Furthermore, the EEG findings in established SE do not necessarily progress through the series of defined stages suggested by some authors.

Treatment of refractory generalized status epilepticus with continuous infusion of midazolam.

The optimal therapeutic approach for the patient with refractory generalized status epilepticus remains to be defined. We describe four patients with refractory generalized status epilepticus who were successfully treated with intravenous midazolam. Each patient had prolonged convulsive status epilepticus unresponsive to standard doses of intravenous benzodiazepines, phenytoin, and phenobarbital. The patients subsequently received midazolam administered as an intravenous bolus (200 micrograms/kg) followed by a continuous infusion (0.75 to 11 micrograms/kg/min) lasting 8 hours to 10 days. Clinical examination and scalp electroencephalographic monitoring documented the cessation of seizure activity within minutes of the loading dose in all patients. No significant adverse effects occurred during midazolam treatment. The one patient with prolonged midazolam infusion required fluid boluses and pressors for moderate hypotension, and the remainder of the patients safely tolerated midazolam despite preexistent hemodynamic instability. All patients recovered and maintained good seizure control. Intravenous midazolam appears to be an effective treatment for refractory generalized status epilepticus, and may represent a substantial improvement over current therapeutic approaches such as pentobarbital anesthesia.

Status epilepticus at an urban public hospital in the 1980s.

We retrospectively reviewed the clinical course of adult patients treated for generalized status epilepticus (SE) at the San Francisco General Hospital (SFGH) from 1980 to 1989. The review was designed to determine whether the etiologies of SE at our hospital have changed over the last two decades, and to investigate the relationships between etiology, response to anticonvulsant therapy, and short-term clinical outcome. Of 154 patients reviewed, the four leading etiologies for SE were anticonvulsant drug withdrawal (39), alcohol-related (39), drug toxicity (14), and CNS infection (12). This pattern was essentially unchanged from observations made at SFGH in the 1970s. Sixty percent of all patients responded to first-line drug treatment (usually phenytoin +/- diazepam), and the remainder required an additional agent (usually phenobarbital) for control of SE. The best response to anticonvulsants occurred in patients with SE related to tumor, anticonvulsant drug withdrawal, or refractory epilepsy, and the poor responders had anoxia, drug toxicity, CNS infection, or other metabolic abnormalities. Seventy-six percent of the patients had good outcomes. Of the 22 patients who died, SE was a likely cause of death in only two (ie, 1.3% of the entire study group). Metabolic abnormalities, stroke, and anoxia were associated with particularly poor outcomes compared with other etiologies. These observations show that the etiologies of SE have remained similar over two successive decades, and that the etiology of SE may help predict both the initial response to drug therapy and the short-term outcome.

Prognostic factors of pentobarbital therapy for refractory generalized status epilepticus.

Pentobarbital coma (PBC) is a treatment for patients with refractory status epilepticus, but there are currently few guidelines for choosing when to initiate or continue this therapy. To identify potential prognostic factors in this setting, we reviewed the course of 17 adult patients treated with a standardized protocol of PBC for refractory status epilepticus over the past 6 years. PBC was extremely effective in aborting seizures in 16 of 17 patients, but 11 of the patients developed severe hypotension that required therapy with vasopressors. Six of the patients had full recoveries or developed only minimal residual deficits following PBC, two developed severe neurologic deficits, and nine died. Survival was associated with a history of epilepsy, absence of multiorgan failure before or during PBC, age < 40 years, and absence of hypotension requiring vasopressors during PBC. Long-term follow-up in seven of eight survivors (mean, 2.9 years; range, 1 to 5 years) showed that patients' conditions remained stable after discharge from the hospital. Thus, although PBC is effective in controlling ongoing seizures, the therapy frequently leads to significant hypotension. This side effect may be especially troublesome in patients with the negative prognostic indicators identified in this study. These findings highlight the need for alternative approaches in the management of these patients.

Barbiturate anesthesia in the treatment of status epilepticus: clinical experience with 14 patients.

We report our experience using barbiturate anesthesia for the treatment of refractory status epilepticus. Following a retrospective review of eight patients treated with a variety of barbiturates and dosing regimens, we established a specific protocol employing pentobarbital and evaluated it prospectively in six patients. Among the 14 patients, intravenous barbiturates, when administered with a loading dose followed by continuous infusion, were uniformly effective in aborting seizures and producing a burst-suppression EEG pattern. Other than the pupillary light reflex, most patients lost all brainstem reflexes and motor responses during therapy. Barbiturate-induced hypotension was observed in 9 of the 14 patients, and required treatment with pressors in seven cases. Three patients died early as a consequence of their underlying illness, while three others died late for reasons unrelated to the status itself or to anticonvulsant therapy. The time to recovery of function following anesthesia varied highly, spanning hours to days for return of motor function and days to weeks for cognition. Of the eight survivors, four were left with mild cognitive deficits, one returned to his baseline dementia, and three had residual encephalopathies (severe in two). We conclude that barbiturate anesthesia is an extremely effective therapy for refractory seizures. However, its use necessitates recognition of untoward cardiovascular responses and prolonged intensive care.

Seizures associated with recreational drug abuse.

We retrospectively identified 49 cases of recreational drug-induced seizures in 47 patients seen at the San Francisco General Hospital between 1975 and 1987. Most patients experienced a single generalized tonic-clonic seizure associated with acute drug intoxication, but 7 patients had multiple seizures and 2 patients developed status epilepticus. The recreational drugs implicated were cocaine (32 cases), amphetamine (11), heroin (7), and phencyclidine (4). A combination of drugs was responsible in 11 cases. Seizures occurred independent of the route of administration, and occurred in both first-time and chronic abusers. Ten patients (21%) reported having had prior seizures, all with a close temporal association with drug abuse. Other than 1 patient who developed prolonged status epilepticus that caused a fixed neurologic deficit, most patients had no obvious short-term neurologic sequelae.

Controlled trial of botulinum toxin injections in the treatment of spasmodic torticollis.

We administered local injections of botulinum toxin to 20 patients with torticollis in a blinded, placebo-controlled study. Each patient received four sets of injections: three different doses of botulinum toxin and one placebo. The order of the sessions was random and unknown to the patients. Sixteen of the patients (80%) reported subjective improvement to at least one dose of botulinum toxin; 11 (55%) reported substantial improvement. No objective benefit was documented. Side effects were minor and transient, although dysphagia occurred in four. Some patients reported that the effect waned despite persistent relaxation or even flaccidity of previously overactive muscles, suggesting a change in the pattern of muscle activity after botulinum toxin injections.