Effect of protein malnutrition on CA3 hippocampal pyramidal cells in rats of three ages

Prenatal and postnatal protein deprivation effect on CA3 hippocamapal pyramidal pyramidal cells were investigated in 30-, and 90- and 220-day old rats Female rats were fed either a 6% or a 25% casein diet 5 wk before conception and the litters were maintained on their respective diet until sacrificed. In 216 rapid Golgi-impregnatd cells, we measured somal size, length and diameter of apical dendrite, number of apical dendrites intersecting 10 concentric rings 38 μm apart, thorny excrescence area and length, head diameter and density of synaptic spines on 50-μm segments of apical dendrite. The present experiments showed that malnutrition produced significant reductions of somal size in animals at 220 days of age. There were significant reductions of apical dendrite diameters in animals of 30 and 90 days, and of density and head diameter of synaptic spines at the three ages studied, and significant decrease of the thorny excrescence area at 220 days of age. At this latter age, dendritic branching was significantly decreased in the last four rings representing the area into which the perforant pathway projects. In 30-day malnourished rats, dendritic branching showed a significant increase in rings 4–6 representing the area in which the Schaffer collaterals synapse. The location of the deficit spines corresponds to the sites where mossy fibers synapse on the apical dendrites of CA3 neurons. Age-related changes normally observed in control rats (e.g., the 30-day-old control group showed the smallest somal size and 220-day-old controls the largest size) failed to occur in the malnourished rats. The deficits in spine density and dendritic branching (in animals of 220 days old) were similar to those found in our previous studies on fascia dentata.     

Variation in electrophysiology and morphology of hippocampal CA3 pyramidal cells

A proportion of pyramidal cells in region CA3 of the mammalian hippocampus generate bursts of action potentials when stimulated with an intracellular injection of depolarizing current. Although a previous study has suggested that burst-type cells are more likely to be located in subregion CA3a than CA3b, it has been unclear if, or how, this burst-type firing was related to cell morphology. In the present study, a sample of pyramidal cells located in subregions CA3a, b and c were recorded intracellularly. Many of these cells were filled with Lucifer yellow, allowing correlation of gross morphology with electrophysiology. Contrary to previous results, it was determined that the proportion of cells which generated bursts did not differ significantly across CA3 subregions. It was found, however, that cells with somata located close to the stratum pyramidale (s.p.)/oriens border ('deep' cells) were more than twice as likely to generate burst-type responses than were cells located closer to stratum radiatum ('shallow' cells). One notable morphological feature of the deep cells was the greater length of the initial portion of their apical dendrite, as measured from soma to primary branching point. This observation is consistent with the hypothesis that burst-type responses are generated or modulated by ion channels on this section of the dendrite.     

Alterations and recovery of dendritic spine density in rat hippocampus following long-term ethanol ingestion

Neuronal loss and dendritic pathology are often observed in humans and animals after long-term ethanol ingestion. It is not known, however, if surviving but damaged neurons can recover normal structure during ethanol abstinence. We quantified dendritic spine density in two neuronal populations in rat hippocampus to investigate whether reversibility from the cellular neurotoxic sequelae of chronic ethanol exposure was possible. Male Long-Evans rats were maintained for 20 weeks on an ethanol-containing liquid diet. Controls were pair-fed a liquid diet with sucrose substituted isocalorically for ethanol. One-half of each group was sacrificed at the end of the 20-week treatment and one-half was given a 20-week ethanol-free recovery period period to sacrifice. Analysis of rapid Golgi material revealed a decreased spine density in CA1 pyramidal cells that increased to control level during abstinence, and an increased spine density in dentate gyrus granule cells that was reduced toward control level during abstinence. Thus, despite the fact that chronic ethanol exposure produced differential initial effects, the return toward normal spine density in each region is consistent with the concept of neuronal recovery and reorganization during abstinence from ethanol.     

Regional sex differences in spine density along the apical shaft of visual cortex pyramids during postnatal development

Dendritic spines from the apical shaft of layer V pyramids were counted on Golgi-stained sections of the monocular subfield of the primary visual cortex of 10-, 20-, 40- and 60-day-old male and female rats. Dendritic segments located in layer IV and at 100–300 μm from the soma had a significantly higher spine content in 10-day-old females when compared to males. This sex difference was extended to outer dendritic segments with increasing age, and became restricted to dendritic segments of outer layers (II–III) located at 400–550 μm from the perikaryon in 40-day-old rats. Sex differences in spine content finally disappear by day 60. These results show the existence of specific laminar and temporal sex differences in the development of dendritic spines in the apical shaft of visual cortex pyramids.     

Dendritic excrescences seem to characterize hippocampal CA3 pyramidal neurons in humans

Summary. Excrescences are unique dendritic postsynaptic structures of the hippocampal formation. Only CA3 pyramidal neurones and hilar mossy cells possess these complex dendritic structures. Dendritic excrescences have so far only been investigated in rabbit, rat and rhesus monkey. Applying a Golgi impregnation method optimized for human brain tissue, we describe the detailed morphology of excrescences of CA3 pyramidal neurons of man. Human thorny excrescences possess a thin and single spine neck and multiple spine heads (4 on average, sometimes more than 10). Human cluster excrescences sit upon the dendrite with a broad stem, and exhibit a “papilloma-like” surface. Some human CA3 pyramidal neurons seem to possess markedly longer spine necks and larger spine heads compared to human neocortical pyramid cells; they were named long-neck spines. Thorny excrescences, cluster excrescences and the newly described long-neck spines can also be found on the dendritic main stem of human CA3 pyramidal neurons. CA2 pyramidal neurons neither possess these long neck spines nor thorny or cluster excrescences. Thus, the unique excrescences of CA3 pyramidal neurones seem to be another criterion for a demarcation between the CA3- and CA2 region of the human hippocampus.     

Adult hippocampal neurogenesis is impaired by transient and moderate developmental thyroid hormone disruption

The hippocampus maintains a capacity for neurogenesis throughout life, a capacity that is reduced in models of adult onset hypothyroidism. The effects of developmental thyroid hormone (TH) insufficiency on neurogenesis in the adult hippocampus, however, has not been examined. Graded degrees of TH insufficiency were induced in pregnant rat dams by administration of 0, 3 or 10 ppm of 6-propylthiouracil (PTU) in drinking water from gestational day (GD) 6 until weaning. Body, brain, and hippocampal weight were reduced on postnatal day (PN) 14, 21, 78 and hippocampal volume was smaller at the 10 but not 3 ppm dose level. A second experiment examined adult hippocampal neurogenesis following developmental or adult onset hypothyroidism. Two male offspring from 0 and 3 ppm exposed dams were either maintained on control water or exposed to 3 ppm PTU to create 4 distinct treatment conditions (Control-Control; Control-PTU, PTU-Control, PTU-PTU) based on developmental and adult exposures. Beginning on the 28th day of adult exposure to 0 or 3 ppm PTU, bromodeoxyuridine (BrdU, 50 mg/kg, ip) was administered twice daily for 5 days, and one male from each treatment was sacrificed 24 h and 28 days after the last BrdU dose and brains processed for immunohistochemistry. Although no volume changes were seen in the hippocampus of the neonate at 3 ppm, thinning of the granule cell layer emerged in adulthood. Developmental TH insufficiency produced a reduction in newly born cells, reducing BrdU + ve cells at 1 with no further reduction at 28-days post-BrdU. Similar findings were obtained using the proliferative cell marker Ki67. Neuronal differentiations was also altered with fewer doublecortin (Dcx) expressing cells and a higher proportion of immature Dcx phenotypes seen after developmental but not adult TH insufficiency. An impaired capacity for neurogenesis may contribute to impairments in synaptic plasticity and cognitive deficits previously reported by our laboratory and others following moderate degrees of developmental TH insufficiency induced by this PTU model.     

Early dendritic changes in hippocampal pyramidal neurones (field CA1) of rats subjected to acute soman intoxication: a light microscopic study

In rats poisoned with soman (s.c. 100 μg/kg), a potent inhibitor of cholinesterase (ChE), the numbers of dendritic spines of Golgi impregnated hippocampal pyramidal cells (CA1 sector) were evaluated within the first hour of the intoxication. Animals that experienced convulsions showed a rapid and striking decrease in the density of dendritic spines which could be reduced by nearly 80% of the controls in the basal dendrites 60 min post-soman exposure. Although the exact mechanisms cannot be determined from the present study, it is suggested that the spine loss may represent: (1) the first sign of the seizure-related neuronal changes which are known to occur later during soman intoxication; and (2) the expression of the ‘dendrotoxic’ effects produced by certain non-cholingergic excitatory transmitters such as glutamate.     

Protein synthesis is necessary for dendritic spine proliferation in adult brain slices

Dendritic spines, small protrusions from dendritic shafts, receive most of the excitatory synapses in cortical regions. Spines are highly plastic structures that can be rapidly produced or lost in response to a wide array of internal and external stimuli, and they proliferate in acute slice preparations [J. Neurosci. 19 (1999) 2876]. The goal of the present study was to determine if protein synthesis is necessary for this spine proliferation. We found that the addition of protein synthesis inhibitors to acute slices (in which spines otherwise proliferate) blocked new spine growth. Furthermore, a population of longer spines was observed after 2 h but these did not develop during protein synthesis blockade. These data suggest that protein synthesis is necessary for new spine growth in acute brain slice preparations and support literature suggesting that newly produced spines develop from filopodia-like protrusions.     

Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat

We have previously shown that the density of dendritic spines on hippocampal CA1 pyramidal cells is dependent on circulating estradiol and progesterone and fluctuates naturally during the 5 day estrous cycle in the adult rat. To date, however, no detailed characterization of the roles that these hormones play in regulation of spine density has been made. In order to determine the time courses and extent of the effects of estradiol and progesterone on dendritic spine density, we have analyzed the density of dendritic spines on the lateral branches of the apical dendritic tree of Golgi-impregnated CA1 hippocampal pyramidal cells in several experiments. In summary, our findings included the following: (1) Following ovariectomy, circulating estradiol is undetectable within 24 hours; however, spine density decreases gradually over a 6 day period. (2) Spine density does not decrease any further up to 40 days following ovariectomy. (3) Treatment with estradiol alone can reverse the ovariectomy-induced decrease in spine density. (4) Spine density begins to increase within 24 hours following estradiol benzoate injection in an ovariectomized animal, peaks at 2 and 3 days, then gradually decreases over the next 7 day period. (5) Although free estradiol is metabolized more rapidly than estradiol benzoate, there is no difference in the rate of decrease in spine density following injection of either form. (6) Progesterone has a biphasic effect on spine density in that progesterone treatment following estradiol initially increases spine density for a period of 2 to 6 hours but then results in a much sharper decrease than is observed following estradiol alone. By 18 hours following progesterone treatment, spine density is decreased nearly to 6 day ovariectomy values. (7) Treatment of intact rats with the progesterone receptor antagonist, RU 486, during the proestrus phase of the estrous cycle inhibits the proestrus to estrus drop in spine density. These findings account for both the gradual increase and rapid decrease in spine density which we have previously observed during the estrous cycle and indicate that progesterone in particular may be an important factor in the regulation of rapid morphologic changes which occur naturally in the adult brain. © 1993 Wiley-Liss, Inc.     

Cholinergic deafferentation enhances rat hippocampal pyramidal neuron responsiveness to locally applied nicotine

We tested whether cholinergic denervation of the hippocampas of young rats would result in an enhancement of CAI pyramidal cell responsiveness to nicotine. Electrolytic lesions of the medial septal area were performed in young male Fisher 344 rats, One month later the rats were anesthetized with pentobarbital and nicotine was locally applied to CAI pyramidal neurons using pressure microejection. The dose of nicotine required to excite the pyramidal neurons was significantly lower for cells recorded from rats with septal lesions. However, no changes in hippocampal cytisine or α-bungarotoxin binding were found.     

Dendritic shrinkage and dye-coupling between rat hippocampal CA1 pyramidal cells in the tetanus toxin model of epilepsy

A small dose of tetanus toxin injected into the rat hippocampus produces a chronic model of temporal lobe epilepsy. We have examined whether morphological changes occur in hippocampal CA1 pyramidal cells in this model by using intracellular injections of biocytin. Eight weeks after the injection of tetanus toxin, significantly more ‘dye-coupled’ cells were found in this group than in the buffer (control) injected group (63% compared with 7%). Half of these coupled cells appeared to be linked at the soma, and the other half by dendrodendritic contacts. Analysis of the dendritic trees revealed that the tetanus toxin group showed a decrease in complexity around the proximal to mid-apical dendritic regions and around the mid- to distal basal dendritic regions. The dye-coupling indicates that electrotonic interaction is induced or strengthened between hippocampal neurones, possibly as a result of the epilepsy-induced dendritic damage.     

Postpubertal decrease in hippocampal dendritic spines of female rats

Hippocampal dendritic spine and synapse numbers in female rats vary across the estrous cycle and following experimental manipulation of hormone levels in adulthood. Based on behavioral studies demonstrating that learning patterns are altered following puberty, we hypothesized that dendritic spine number in rat hippocampal CA1 region would change postpubertally. Female Sprague-Dawley rats were divided into prepubertal (postnatal day (P) 22), peripubertal (P35) and postpubertal (P49) groups, with the progression of puberty evaluated by vaginal opening, and estrous cyclicity subsequently assessed by daily vaginal smears. Spinophilin immunoreactivity in dendritic spines was used as an index of spinogenesis in area CA1 stratum radiatum (CA1sr) of hippocampus. First, electron microscopy analyses confirmed the presence of spinophilin specifically in dendritic spines of CA1sr, supporting spinophilin as a reliable marker of hippocampal spines in young female rats. Second, stereologic analysis was performed to assess the total number of spinophilin-immunoreactive puncta (i.e. spines) and CA1sr volume in developing rats. Our results indicated that the number of spinophilin-immunoreactive spines in CA1sr was decreased 46% in the postpubertal group compared to the two younger groups, whereas the volume of the hippocampus underwent an overall increase during this same developmental time frame. Third, to determine a potential role of estradiol in this process, an additional group of rats was ovariectomized (OVX) prepubertally at P22, then treated with estradiol or vehicle at P35, and spinophilin quantified as above in rats perfused on P49. No difference in spinophilin puncta number was found in OVX rats between the two hormone groups, suggesting that this developmental decrease is independent of peripheral estradiol. These changes in spine density coincident with puberty may be related to altered hippocampal plasticity and synaptic consolidation at this phase of maturity.     

Constitutively enhanced p21Ras activity amplifies dendritic remodeling of hippocampal neurons during physical activity

In the present study we show that overexpression of constitutively active Ras amplifies the dendritic remodeling observed when animals were allowed to be physically active. The monomeric G-protein Ras is a key molecular trigger of distinct signal transduction pathways that play an important role in proper functioning of neurons. Our previous studies on Ras-transgenic synRas mice have demonstrated a considerable impact of Ras on dendritic growth, extension and synaptic connectivity of neurons. Voluntary access to a running wheel resulted in enlargement of hippocampal pyramidal cell dendrites in wild-type mice as expected. However, constitutively elevated Ras activity further enhanced dendritic growth and branching especially of apical arbors. The resultant dendritic surface gain was paralleled by a significant increase in dendritic spine density. Since Ras is crucially involved in signaling and cascades of neurotrophins that are elevated after physical activity, these results strongly suggest an important role of Ras in dendritic dynamics during induced neuronal remodeling.     

Dendritic action potentials activated by NMDA receptor-mediated EPSPs in CA1 hippocampal pyramidal cells

Intradendritic recordings were obtained in rat CA1 hippocampal pyramidal cells. Repetitive stimulation produced substantial short-term potentiation of the dendritic excitatory postsynaptic potential (EPSP) which was partly attributable to activation ofn-methyl-d-aspartate receptors. Accompanying the potentiated synaptic response were Na⁺-mediated spikes which appeared to originate at multiple sites in the dendritic arbor. These discrete dendritic action potentials are rarely distinguishable in somatic recordings but may contribute to the subthreshold response at the pyramidal cell body. In addition, dendritic spikes may interact with other voltage-dependent dendritic conductances.     

Gonadal hormones are responsible for maintaining the integrity of spine synapses in the CA1 hippocampal subfield of female nonhuman primates

It is well established that gonadal hormonal manipulation results in morphologic changes in the rat hippocampus. The great similarities in the hippocampal formation between nonhuman primates and humans, as well as the differences in this structure between humans and rats, led to this investigation of whether hormonal manipulation in female subhuman primates influences pyramidal cell spine density in the CA1 hippocampal subfield, as it does in rats. African green monkeys (Cercopithecus aethiops sabaeus) were ovariectomized, and half of the animals received estrogen replacement therapy. One month later, the monkeys were killed. In the first group of experiments, pyramidal cell spines were analyzed on Golgi-impregnated material taken from the CA1 hippocampal subfield. In the second experiment, unbiased electron microscopic stereologic calculations were performed to estimate the volumetric density of spine synapses in the same hippocampal subfield. Analysis of the Golgi-impregnated material showed that the spine density of CA1 pyramidal cells is much lower in the ovariectomized animals than in ovariectomized and estrogen-replaced monkeys. The unbiased, electron microscopic, stereologic calculation confirmed the light microscopic observation. The volumetric density (number of spine synapses/microm(3)) of spine synapses was significantly lower (43.33%) in the ovariectomized animals than in ovariectomized and estrogen-replaced monkeys. Because the hippocampus is involved in specific mnemonic functions, this observation highlights the importance of hormone replacement therapy in postmenopausal conditions.     

Effects of GABA on CA3 pyramidal cell dendrites in rabbit hippocampal slices

Using the in vitro rabbit hippocampal slice preparation, we have investigated the effects of gamma-aminobutyric acid (GABA) iontophoresis on CA3 pyramidal cell dendrites. The predominant response (70% of the cells tested) was a hyperpolarization associated with a 30% decrease in cell input resistance (Rm). These hyperpolarizations displayed a very pronounced voltage dependency: they were decreased by cell depolarization and flattened by hyperpolarization. Bicuculline methiodide (BMI, 50 microM) did not abolish this response, nor did intracellular iontophoresis of chloride ions. In 5% of the cells, an additional hyperpolarization was obtained with longer ejection times; it reversed close to the reversal potential of the early component of the IPSP. In 25% of the cells, dendritic GABA application produced a depolarization. This response was reversed with cell membrane depolarization and was associated with a large (80%) decrease in Rm. The depolarizations were abolished by BMI (50 microM) and greatly increased by increasing the intracellular chloride concentration. None of the responses to GABA were affected by blockade of synaptic transmission. We conclude that the predominant response of CA3 pyramidal cell dendrites to GABA application is a hyperpolarization mediated by GABAB receptors and probably carried by potassium ions. The depolarizing responses are mediated via GABAA receptors and depend on an increase in chloride permeability.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Coupling in rat hippocampal slices: Dye transfer between CA1 pyramidal cells

Intracellular injections of Lucifer Yellow-CH (LY) into CA1 pyramidal cells were made in rat hippocampal slices to study dye transfer between neurons as evidence that these cells are electrotonically coupled. Extensive control procedures were performed which substantially reduced inadvertent staining. Over half of the neurons were dye-coupled after injections in stratus pyramidale. Dye coupling occurred even when spike amplitudes were greater than or equal to 70 mV throughout the impalement and was still present after chemical synapses were blocked with a low Ca2+ solution containing Mn2+. Somata of dye-coupled cells were usually located within 35 micrometers (post-fixation) of the injected cell and showed no preferred orientation. Fast prepotentials and dye coupling occurred independently. Neurons in superior cervical ganglia, which were sliced and injected using similar procedures, showed no dye coupling. Intradendritic injections of LY in stratum radiatum also yielded dye coupling between CA 1 pyramidal cells, although the dye coupling was less frequent. Within stratum radiatum, neither extracellular ejections nor intracellular injections of interneurons were associated with multiple staining. Thus, injection of LY into the soma or dendrite of a single CA1 pyramidal cell often resulted in multiple staining, and in many ensembles the somata were well spaced. Control experiments suggested that such dye transfer is not by an extracellular route. This implied that some CA1 cells are electrotonically coupled. Further electrophysiological and morphological studies are required to resolve the discrepancies among various techniques used to evaluate the amount of coupling in the hippocampus.     

Dendritic development of hippocampal CA1 pyramidal cells in a neonatal hypoxia–ischemia injury model

It is believed that neonatal hypoxia–ischemia (HI) brain injury causes neuron loss and brain functional defects. However, the effect of HI brain injury on dendritic development of the remaining pyramidal cells of the hippocampus and the reaction of contralateral hippocampal neurons require further studies. The Morris water maze and Golgi–Cox staining were used to evaluate the learning and memory and dendritic morphology of pyramidal cells. The results of Golgi–Cox staining showed CA1 pyramidal neurons of HI injury models with fewer bifurcations and shorter dendrite length than the naive control group. The density of dendritic spines of hippocampal CA1 pyramidal neurons was significantly lower in the HI brain injury group than in controls. With respect to hippocampal function, the HI brain injury group presented cognitive deficits in the reference memory task and probe trail. In the HI group, the pyramidal cells of left hippocampus that did not experienced ischemia but did experience hypoxia had more complex dendrites and higher density of spine than the HI injury side and control. The functional implementation of injured hippocampus might depend mainly on the hypertrophy of contralateral hippocampus after HI brain injury. Corticosterone can partially prevent the hippocampal pyramidal cells from HI injury and reduce the difference of the bilateral hippocampus pyramidal cells, but there was no improvement in learning and memory. © 2013 Wiley Periodicals, Inc.