Dcdc2 knockout mice display exacerbated developmental disruptions following knockdown of doublecortin.

The dyslexia-associated gene DCDC2 is a member of the DCX family of genes known to play roles in neurogenesis, neuronal migration, and differentiation. Here we report the first phenotypic analysis of a Dcdc2 knockout mouse. Comparisons between Dcdc2 knockout mice and wild-type (wt) littermates revealed no significant differences in neuronal migration, neocortical lamination, neuronal cilliogenesis or dendritic differentiation. Considering previous studies showing genetic interactions and potential functional redundancy among members of the DCX family, we tested whether decreasing Dcx expression by RNAi would differentially impair neurodevelopment in Dcdc2 knockouts and wild-type mice. Consistent with this hypothesis, we found that deficits in neuronal migration, and dendritic growth caused by RNAi of Dcx were more severe in Dcdc2 knockouts than in wild-type mice with the same transfection. These results indicate that Dcdc2 is not required for neurogenesis, neuronal migration or differentiation in mice, but may have partial functional redundancy with Dcx.

Enhanced auditory reversal learning by genetic activation of protein kinase C in small groups of rat hippocampal neurons.

The hippocampus has a central role in specific types of learning, but there is only limited evidence identifying the requisite molecular changes in ensembles of hippocampal neurons. To investigate the role of protein kinase C (PKC) pathways in hippocampal mediated learning, a constitutively active, catalytic domain of rat PKC betaII was delivered into hippocampal dentate granule neurons using a Herpes Simplex Virus (HSV-1) vector. This PKC causes a long-lasting, activation-dependent increase in neurotransmitter release from cultured cells. Activation of PKC pathways in a small percentage (< or =0.26%) of dentate granule neurons was sufficient to enhance rat auditory discrimination reversal learning. The affected neurons altered hippocampal physiology as revealed by elevated NMDA receptor densities in specific hippocampal areas. Thus, these results directly suggest that activation of PKC pathways in a specific hippocampal area alters rat auditory discrimination reversal learning. Because each rat may contain a unique pattern of affected neurons, there appears to be considerable flexibility and/or redundancy in the groups of neurons that can modify learning.

Altered interneuron development in the cerebral cortex of the flathead mutant.

One approach to defining mechanisms essential to neocortical development is to analyze the phenotype of novel spontaneous mutations that dramatically affect the generation and differentiation of different neocortical neurons. Previously we have shown that there is a large decrease in the total number of cortical neurons in the flathead mutant rat, and in this paper we show that the flathead (fh/fh) mutation causes an even larger decrease in the number of interneurons. The decrease in relative interneuron number is different in different cortical lamina and for different interneuron subtypes. Specifically, the percentage of GABA and calretinin- positive cells in upper layers of somatosensory cortex is not appreciably decreased in homozygous mutants, while other interneuron subtypes in somatosensory cortex and all GABA-positive interneuron types in entorhinal cortex are decreased. In addition, the soma and dendritic arbors of interneurons in flathead are greatly hypertrophied, while those of pyramidal neurons are not. Furthermore, we found that at embryonic day 14, flathead mutants display high levels of cell death throughout both the neocortical and ganglionic eminence (GE) proliferative zones with a larger increase in cell death in the GE than in the neocortical VZ. In addition, we provide evidence that there is widespread cytokinesis failure resulting in binucleate pyramidal cells and interneurons, and the number of binucleate interneurons is greater than the number of binucleate pyramidal neurons. Together, these results demonstrate that the fh mutation causes dramatic alterations in interneuron development, and suggest that the flathead mutation causes differential cytokinesis failure and cell death in different types of neocortical progenitors.

Protective effects of prenatal choline supplementation on seizure-induced memory impairment.

Choline is an essential nutrient for rats and humans, and its availability during fetal development has long-lasting cognitive effects (Blusztajn, 1998). We investigated the effects of prenatal choline supplementation on memory deficits associated with status epilepticus. Pregnant rats received a control or choline-supplemented diet during days 11-17 of gestation. Male offspring [postnatal day 29 (P29)-32] were tested for their ability to find a platform in a water maze before and after administration of a convulsant dose of pilocarpine at P34. There were no differences between groups in water maze performance before the seizure. One week after status epilepticus (P41-P44), animals that had received the control diet prenatally had a drastically impaired performance in the water maze during the 4 d testing period, whereas prenatally choline-supplemented rats showed no impairment. Neither the seizures nor the prenatal availability of choline had any effect on hippocampal choline acetyltransferase or acetylcholinesterase activities. This study demonstrates that prenatal choline supplementation can protect rats against memory deficits induced by status epilepticus.

Consequences of recurrent seizures during early brain development.

It is well documented that prolonged seizures (status epilepticus) can cause neuronal injury and result in synaptic reorganization in certain brain regions. However, the effect of recurrent, relatively short seizures in young animals on subsequent brain development is not known. To study the consequences of recurrent seizures on the developing brain, we subjected immature rats to a total of 50 flurothyl-induced seizures from postnatal day 11 until day 23. Immunohistochemistry for c-fos was performed to characterize the pattern of neuronal activation following the seizures. Cell counting of dentate granule cells, CA3, CA1, and hilar neurons, using unbiased stereological methods, and the silver impregnation method were used to evaluate neuronal death following the recurrent seizures. Timm and Golgi staining were performed four weeks after the 50th seizure to evaluate the effects of recurrent seizures on synaptic organization. Our results show that recurrent flurothyl-induced seizures progressively increased excitability of the brain, as revealed by a dramatic increase in the extent and intensity of c-fos immunostaining. While no cell loss was detected in the hippocampus with either Cresyl Violet or silver stains, animals experiencing multiple daily seizures developed increased mossy fiber sprouting in both the supragranular layer of the dentate gyrus and the infrapyramidale layer of the CA3 region. Golgi staining confirmed that there was an increase in mossy fibers in the pyramidal cell layer. Our results suggest that serial recurrent seizures in the immature brain can lead to significant changes in mossy fiber distribution even though the seizures do not cause significant hippocampal cell loss.

Effects of hyperthermia and continuous hippocampal stimulation on the immature and adult brain.

Whether febrile seizures lead to hippocampal necrosis is a question of paramount clinical importance. This study attempted to simulate a complex febrile seizure, compared with hyperthermia (HYP) alone and prolonged seizure alone (produced by continuous hippocampal stimulation (CHS)). Four groups of rats were studied at each of two ages, immature (postnatal day, P20) and adult (P60). Group 1 was subjected to 45 min of HYP (body temperature 40 degrees C) plus CHS, Group 2 received 45 min of HYP alone, Group 3 got 45 min of CHS alone, and Group 4 was sham-handled control rats. Baseline and post-session EEGs were recorded in all groups. Subsequently, brains were examined histologically for evidence of hippocampal damage. Both CHS-treated groups (with and without HYP) exhibited behavioral and EEG seizures while the group undergoing HYP alone did not have seizures. There were no gross histological lesions in any group. Cell counts in regions CA1, CA3, dentate gyrus and dentate hilus did not differ in rats under any condition of hyperthermia and CHS, in either P20 or P60 rats compared to age-matched controls. These results indicate that both immature and mature rodents are resistant to hyperthermic brain damage and raises the question of whether febrile seizures play a role in the genesis of mesial temporal sclerosis.

Characterization of seizures in the flathead rat: a new genetic model of epilepsy in early postnatal development.

PURPOSE: Disorders in normal central nervous system (CNS) development are often associated with epilepsy. This report characterizes seizures in a novel genetic model of developmental epilepsy, the Flathead (FH) rat. METHODS: Animals (n = 76) ages P0-22 were monitored for clinical and electrographic seizure activity. The effects of various AEDs on seizure frequency and duration also were assessed: phenobarbital (PB; 40 mg/kg), valproate (VPA; 400 mg/kg), or ethosuximide (ESM; 600 mg/kg). RESULTS: FHs display episodes of behavior characterized by whole-body tremor, strub tail, alternating forelimb clonus, and complete tonus. EEG recordings from neocortex reveal that FH seizures are bilateral and begin around P7. Seizures occur at a frequency of approximately six per hour from P7 to P18 and the average duration of seizures increases through development. PB, VPA, and ESM failed to prevent seizures; however, PB significantly increased the interval of seizures but had no effects on the duration of seizures, whereas VPA decreased the duration of seizures and not the interval. CONCLUSIONS: Seizures in FH rats occur at a constant and high frequency through a defined period in early postnatal development, and these seizures are not completely blocked by high doses of PB, VPA, or ESM. Because FH is a single-locus mutant displaying a highly regular pattern of seizure activity, it is an ideal model for examining the process of epileptogenesis in the developing brain, evaluating new AED therapies, and determining the identity of a gene essential to the normal development of cortical excitability.

A gene essential to brain growth and development maps to the distal arm of rat chromosome 12.

A recently discovered, spontaneous, autosomal recessive mutation in rats, flathead (fh), results in greatly reduced brain growth beginning in late fetal development. In this study we have mapped the fh mutation by determining the pattern of segregation of polymorphic microsatellite markers with respect to fh in 51 affected F2 offspring from a single interstrain intercross. Two markers on chromosome 12, D12Rat80 and D12Mgh6, cosegregated with the fh mutation in all 51 affected animals. The distribution of six additional markers in 40 informative meioses further localizes fh approximately 2 cM teleomeric to nos1. There are no known mutations in homologous regions of either mouse or human genomes that result in deficits in late neurodevelopment similar to that observed in fh/fh animals. The unique phenotype of fh/fh animals and the location of fh suggests the presence of a novel gene essential to normal brain development on the distal end of rat chromosome 12.

Synaptic reorganization following kainic acid-induced seizures during development.

Prolonged seizures in the adult brain causes neuronal loss in the hippocampus and aberrant growth (sprouting) of granule cell axons (mossy fibers) in the supragranular zone of the fascia dentata and stratum infrapyramidale of CA3. There is considerable evidence that these changes in neuronal growth following seizures are age related, with younger animals having fewer reactive changes following prolonged seizures than older animals. However, there is little information available regarding the age at which seizures in the developing brain result in alterations in axonal growth and synapse formation. In this study, we evaluated the effects of kainic acid (KA)-induced seizures during development on synaptic reorganization using the expression of growth-associated protein-43 (GAP-43), a marker for synaptogenesis and Timm stain which detects the presence of zinc in granule cell axons. Age specific doses of KA were used to induce seizures of similar intensity at various ages (postnatal days (P) 12, 21, 25, 35, 45, 60) in Sprague-Dawley rats. Up to the age of P25, there were no differences in either Timm or GAP-43 staining between animals with KA seizures and controls. In P25 and older KA-treated rats, Timm staining was found in the supragranular layer of the dentate gyrus. This staining increased with age at the time of KA injection. Seizures in adult (P60), but not younger rats also resulted in increased staining in the suprapyramidal layer of the CA3 subfields. Changes in GAP-43 were delayed compared to the Timm staining with no differences between KA-treated animals and controls until P35 when a band of GAP-43 immunostaining appeared in the supragranular inner molecular layer, progressively increasing in intensity and thickness with time. This study demonstrates that seizure-induced reactive synaptogenesis is age-related. Since both Timm and GAP-43 reflect different aspects of reactive synaptogenesis, used in combination these methods provide useful information about the structural changes following seizures during development.

Seizure-induced glutamate release in mature and immature animals: an in vivo microdialysis study.

A glutamate biosensor was used to measure extracellular glutamate concentrations in the hippocampus of mature and immature animals. Significant elevations of extracellular glutamate were observed following seizures induced by either kainic acid or pilocarpine. The degree of glutamate increase following seizures was similar in both mature and immature animals. These results suggest that excitotoxicity may play a role in seizure-induced brain damage in the adult brain. In the immature brain, however, no brain damage is seen after seizures, suggesting that glutamate release may not cause as significant excitotoxic damage early in development.

[Consequences of recurrent seizures during development]. / Consecuencias de la epilepsia refractaria durante el desarrollo.

While many children with recurrent seizures have a good prognosis, a small percentage of children with intractable epilepsy have a more ominous course with a gradual decline in cognitive abilities over time. While the reasons for this cognitive decline may be multifactorial, there is evidence both from human and animal studies that recurrent seizures may lead to gradual cognitive impairment in some children. Laboratory studies have also demonstrated that recurrent seizures can lead to deficits in learning and memory as well as structural changes in the brain. It is important for the clinical to be aware of gradual declines in intelligence that may occur over time.

Multiple kainic acid seizures in the immature and adult brain: ictal manifestations and long-term effects on learning and memory.

PURPOSE: While there is increasing evidence that the adverse effects of prolonged seizures are less pronounced in the immature than in the mature brain, there have been few investigations of the long-term effects of recurrent seizures during development. This study examined the effects of multiple administrations of the convulsant kainic acid (KA) on seizure characteristics and spatial learning as a function of brain development. METHODS: To determine the long-term effects of serial KA seizures during ontogeny, saline or convulsant doses of KA were given intraperitoneally 4 times, at 2-day intervals. Immature rats were given KA on P20, P22, P24 and P26; adult rats got KA on P60, P62, P64 and P66. Ictal characteristics and EEGs were recorded. To examine the effects of multiple KA seizures on the retention of spatial learning, water maze testing was performed before (immature group: from P16-19, adult group: from P56-P59) and after (immature: from P60-P63, adult: from P100-P103) KA injections. Finally, histology was performed to compare KA-induced damage at each age. RESULTS: In immature animals, serial KA administration resulted in seizures with a progressively longer onset latency and decreased severity. In contrast, KA serially administered to adult rats caused severe seizures after each of the 4 injections. In immature rats, epileptiform EEG changes were most prominent after the first KA injection, whereas in adults, prolonged paroxysmal EEG patterns were seen after all 4 KA injections. Before KA, both rat pups and adults acquired place learning in the water maze. One month after the final KA injection, there was no deficit in spatial learning retention in the immature group, whereas the adult group had profound impairment compared to age-matched, saline-injected controls. Histology revealed no lesions in immature rats treated multiple times with KA but profound cell loss in hippocampal fields CA4, CA3 and CA1 in rats treated serially with KA as adults. CONCLUSIONS: Previous studies have shown that a single KA injection causes prolonged status epilepticus (which persists for several hours), leading to severe histologic and behavioral sequelae in adult rats but not in pups. Our study extends those findings, demonstrating that immature rats are spared the cognitive and pathological sequelae of multiple injections of convulsant doses of KA as well.