Hippocampal Subareas Research Articles

Developmental changes in the expression of iron regulatory proteins and iron transport proteins in the perinatal rat brain

The perinatal brain requires a tightly regulated iron transport system. Iron regulatory proteins (IRPs) 1 and 2 are cytosolic proteins that regulate the stability of mRNA for the two major cellular iron transporters, transferrin receptor (TfR) and divalent metal transporter-1 (DMT-1). We studied the localization of IRPs, their change in expression during perinatal development, and their relationship to TfR and DMT-1 in rat brain between postnatal days (PND) 5 and 15. Twelve-micron frozen coronal sections of fixed brain tissue were obtained from iron-sufficient Sprague-Dawley rat pups on PND 5, 10, and 15, and were visualized at 20 to 1,000x light microscopy for diaminobenzidine activity after incubation with specific primary IRP-1, IRP-2, DMT-1, and TfR antibodies and a universal biotinylated secondary and tertiary antibody system. IRP and transport protein expression increased in parallel over time. IRP1, IRP2, and DMT-1 were partially expressed in the choroid plexus epithelial cells at PND 5 and 10, and fully expressed at PND 15. The cerebral blood vessels and ependymal cells strongly expressed IRP1, IRP2, and DMT-1 as early as PND 5. Substantive TfR staining was not seen in the choroid plexus or ependyma until PND 15. Glial and neuronal expression of IRP1, IRP2, DMT-1, and TfR in cortex, hippocampal subareas and striatum increased over time, but showed variability in cell number and intensity of expression based on brain region, cell type, and age. These developmental changes in IRP and transporter expression suggest potentially different time periods of brain structure vulnerability to iron deficiency or iron overload.

A grading system for hippocampal sclerosis based on the degree of hippocampal mossy fiber sprouting.

In patients suffering from temporal lobe epilepsy (TLE) a highly variable degree of hippocampal sclerosis (HS) can be observed. For standard neuropathological evaluation after hippocampal resection, neuronal cell loss in the hippocampal subareas is assessed (Wyler score 0-4) [Wyler et al. (1992) J Epilepsy 5: 220-225]. Other marked morphological changes in the sclerotic hippocampus are gliosis and loss of mossy fibers in the hilus and mossy fiber sprouting in the supragranular layer. In this study we quantified changes in mossy fiber density using Timm's stain in resected hippocampal tissue from patients with various degrees of sclerosis. We found that tissue specimens from patients without sclerosis (W0) show almost no mossy fiber sprouting. Patients with moderate sclerosis show sprouting without fiber loss in the hilus, whereas specimens from patients with severe sclerosis show sprouting as well as fiber loss in the hilus. Thus, analysis of mossy fiber abundance in hilus and supragranular layer by the rapid and simple Timm's stain is a sensitive measure for hippocampal sclerosis. It provides a reliable rapid tool for neuropathological evaluation, even if the tissue only contains dentate gyrus due to the sectioning procedure.

Perinatal Brain Iron Deficiency Increases the Vulnerability of Rat Hippocampus to Hypoxic Ischemic Insult

Fetal brain iron deficiency occurs in human pregnancies complicated by diabetes mellitus or intrauterine growth retardation. Because neurocognitive deficits are more common in the offspring of these pregnancies, we tested the hypothesis that perinatal brain iron deficiency predisposes the neonatal hippocampus, a structure important for memory processing, to injury. Brain iron concentration was reduced by 45% in 45 neonatal rats by maternal dietary iron restriction during gestation. Right-sided neuronal injury in four hippocampal subareas was induced by hypoxic-ischemic insult (ipsilateral carotid artery ligation and subsequent hypoxia on postnatal d 7) and was quantified histochemically on d 8 by cytochrome c oxidase activity (n = 30), and on d 14 by Nissl staining (n = 15). Acute right-sided cytochrome c oxidase activity loss occurred in CA1 (P = 0.02), CA3c (P < 0.001) and dentate gyrus (P < 0.001) in the iron-deficient group, whereas only CA1 (P = 0.003) was affected in the iron-sufficient group. Long-term right-sided Nissl substance loss occurred in CA1 (P = 0.001), CA3a,b (P < 0.001) and dentate gyrus (P = 0.008) in the iron-deficient group, but only in CA1 (P = 0.004) in the iron-sufficient group. No increase in right-sided free-iron staining was present in either group. Perinatal iron deficiency predisposes the neonatal hippocampus to a greater acute loss of neuronal metabolic activity after an hypoxic-ischemic event, suggesting compromised cellular energetics. The subsequently greater loss of hippocampal neuronal integrity suggests poorer recoverability after injury in the perinatal iron-deficient brain.

Perinatal Brain Iron Deficiency Predisposes the Rat Hippocampus to Long-term Damage following Hypoxic-ischemic Insult • 1566

The offspring of pregnancies complicated by diabetes mellitus or intrauterine growth retardation are at increased risk for perinatal asphyxia and for long-term neurocognitive sequelae. Both gestational conditions are characterized by an increased prevalence of brain iron deficiency. The hippocampus is important for a variety of cognitive functions (e.g., recognition memory) and is susceptible to hypoxicischemic (HI) injury. To evaluate the hypothesis that brain iron deficiency increases the vulnerability of the hippocampus to HI injury and to assess whether iron induced reperfusion injury has any role in such injuries, we ligated the right common carotid artery in 15 perinatally iron-deficient and 15 iron-sufficient 7-day-old rat pups,and exposed them to 2.5 hours of 8% FIO2(Levine model). The non-ligated left side served as the undamaged internal control. On day 14, neuronal injury was quantified histochemically by assessing intensity of staining for Nissl substance in 4 hippocampal subareas in both hemispheres using optical densitometry in gray scale units (range: 1/white-254/black). To assess iron induced reperfusion injury, sections were stained for iron by modified Perl's reaction. The brain iron concentration of the iron-deficient group was 55% of control (p<0.001). In the iron-deficient group,there was significant loss of Nissl substance on the ligated right side in subareas CA1 (124 ± 14 v. 112 ± 15, p=0.002), CA3ab (144 ± 10 v. 129 ± 15, p=0.001) and the dentate gyrus (136 ± 14 v. 128 ± 13, p=0.012), when compared with the unligated left side.The iron-sufficient group had significant right sided Nissl loss only in CA1 (111 ± 14 v. 106± 15, p = 0.05). The mean number of subareas with detectable right sided Nissl loss per animal was greater in the iron-deficient group than the iron-sufficient group (2.9 ± 1.0 v. 2.0 ± 1.2, p<0.05). The degree of right-sided Nissl substance loss per animal in the iron-deficient group was also greater in CA1(p=0.042), CA3ab(p=0.003) and the dentate gyrus(p=0.037)when compared with the iron-sufficient group. There were no side-to-side differences in the number of iron stained cells in either group. Perinatal iron deficiency increases the vulnerability of hippocampal subareas of CA1, CA3ab and dentate to HI injury. Iron deposition is not involved in the pathophysiology of this injury. These findings may explain the increased risk of perinatal asphyxia and its neurocognitive sequelae in infants born of pregnancies complicated by perinatal iron deficiency.

Changes in voltage-dependent calcium channel α 1-subunit mRNA levels in the kindling model of epileptogenesis

The establishment of a focus of epileptiform activity in the hippocampus of the rat, using the kindling paradigm, leads to enhanced voltage-dependent calcium conductance of CA1 pyramidal neurones (G.C. Faas, M. Vreugdenhil, W.J. Wadman, Calcium currents in pyramidal CA1 neurones in vitro after kindling epileptogenesis in the hippocampus of the rat, Neuroscience 75 (1996) 57–67; M. Vreugdenhil, W.J. Wadman, Kindling-induced long-lasting enhancement of calcium in hippocampal CA1 area of the rat: relation to calcium-dependent inactivation, Neuroscience 59 (1994) 105–114). Using semi-quantitative in situ hybridization techniques, we investigated whether these changes were associated with an altered expression of the genes that encode for the α 1 A–E-subunits of the voltage-dependent calcium channels (VDCC). Kindling epileptogenesis was induced in rats that received an electrical tetanic stimulation of the Schaffer collateral/commissural fibre pathway in the hippocampus twice daily. Two groups of rats were studied before the appearance of generalized seizures, one group after at least 5 generalized seizures (fully kindled) and one group was investigated at long-term (28 days) after the last seizure. During the initial stages of epileptogenesis, the α 1A-, α 1D- and α 1E-subunit mRNA levels were significantly increased in the different hippocampal subareas in comparison to the levels in control animals. In contrast, α 1B-subunit gene expression decreased in the CA1 area and dentate gyrus. No significant change was observed in the α 1C-I and α 1C-II expression. At the fully kindled stage, the only significant change was an up-regulation of the α 1B-subunit mRNA levels in the CA3 area, 24 h after the last seizure. No change in VDCC α 1-subunit gene expression was found in animals investigated long-term after the establishment of the fully kindled state. Thus, the VDCC a 1-subunit gene expression is altered in a subclass-specific manner during the early stages of kindling and may play a role in the establishment of a kindled focus, possibly caused by an alteration of the population of VDCCs involved in neurotransmitter release. The absence of long-lasting changes suggests that the maintenance of a kindled focus is not due to persisting alterations in VDCC α 1 mRNA levels.

An olfactory input to the hippocampus of the cat: Field potential analysis

Hippocampal responses to electrical stimulation of the prepyriform cortex in the cat were studied both in acute experiments under halothane anesthesia and in awake cats with chronically indwelling electrodes. Analysis of field potentials and unit activity indicated the extent to which different hippocampal subareas were activated, the laminar level at which the synaptic action took place and the dynamics of the evoked responses. It was found that: (1) the main generator of evoked responses in the hippocampus upon prepyriform cortex stimulation is localized in the fascia dentata and CA3 (CA1 pyramidal cells, and probably also subiculum cells, are activated but in a lesser degree); (2) the initial synaptic activity takes place at the most distal part of the dendrites of fascia dentata granule cells and CA3 pyramidal cells; and (3) this synaptic activity corresponds to an EPSP that leads to a transient increase in the firing rate of the hippocampal units, which is often followed by a long-lasting decrease in firing rate. We conclude that the pathway from the prepyriform cortex via lateral entorhinal cortex to hippocampal neurons may enable olfactory inputs to effectively excite hippocampal neurons.

Effect of lesions in hippocampal subareas on rat shuttle behavior

Rats with lesions in subareas CA3 and CA4/dentate gyrus of the dorsal hippocampus were submitted to four different experimental situations in a shuttlebox. Animals with CA3 lesions made less shuttle responses to a buzzer than sham-operated or intact control animals in two of the tests, in which the buzzer was paired (i.e., given contiguous) to a footshock: a Pavlovian paradigm, and a typical two-way avoidance situation. Animals with lesions in CA4/dentate gyrus showed no difference in their performance of shuttle responses to the buzzer in any of the four tests. The results suggest that CA3 plays a role in the establishment of stimulus-stimulus relations in the brain, and that CA4 and the dentate gyrus play no major role in any of the aversive situations examined in this paper.