Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus

The immature brain is prone to seizures but the underlying mechanisms are poorly understood. We explored the hypothesis that increased seizure susceptibility during early development is due to the excitatory action of GABA. Using noninvasive extracellular field potential and cell-attached recordings in CA3 of Sprague-Dawley rat hippocampal slices, we compared the developmental alterations in three parameters: excitatory actions of GABA, presence of the immature pattern of giant depolarizing potentials (GDPs) and severity of epileptiform activity generated by high potassium. The GABA(A) receptor agonist isoguvacine increased firing of CA3 pyramidal cells in neonatal slices while inhibiting activity in adults. A switch in the GABA(A) signalling from excitation to inhibition occurred at postnatal day (P) 13.5 +/- 0.4. Field GDPs were present in the form of spontaneous population bursts until P12.7 +/- 0.3. High potassium (8.5 mm) induced seizure-like events (SLEs) in 35% of slices at P7-16 (peak at P11.3 +/- 0.4), but only interictal activity before and after that age. The GABA(A) receptor antagonist bicuculline reduced the frequency or completely blocked SLEs and induced interictal clonic-like activity accompanied by a reduction in the frequency but an increase in the amplitude of the population spikes. In slices with interictal activity, bicuculline typically caused a large amplitude interictal clonic-like activity at all ages; in slices from P5-16 rats it was often preceded by one SLE at the beginning of bicuculline application. These results suggest that, in the immature hippocampus, GABA exerts dual (both excitatory and inhibitory) actions and that the excitatory component in the action of GABA may contribute to increased excitability during early development.     

Developmental changes in synaptic properties in hippocampus of neonatal rats

The properties of synaptic responses in area CA1 of hippocampus were analyzed in slices prepared from 7-9 and 12-15 day old neonate rats. As expected from earlier work, only slices of two-week-old animals showed a consistent degree of long-term potentiation (LTP) in response to patterned high frequency stimulation. Several other synaptic properties were found to change during this developmental period. Inhibitory responses were absent in 7-9 day old but not in 12-15 day old neonates. Paired-pulse facilitation and the calcium sensitivity of postsynaptic responses were considerably reduced in 7-9 as compared to 12-15 day old rats. However, phorbol esters and 4-aminopyridine treatment still produced a strong facilitation of field potentials. The N-methyl-D-aspartate (NMDA) component of responses to single pulse stimulation in low magnesium medium was found to be larger in slices of 7-9 than 12-15 day old or adult animals. At the two time periods examined, trains of high frequency stimulation applied in the presence of regular magnesium elicited an NMDA dependent response. It is concluded that the differences in synaptic properties observed between 7-9 and 12-15 day old neonates may not account for the absence of LTP in the younger animals.     

Developmental changes of MAP2 immunoreactivity in the hippocampus proper and dentate gyrus of the rat

Microtubules are present in high concentration in the nervous system and are a prominent component of the neuronal cytoskeleton. Microtubules are composed of tubulin and variety of microtubule-associated proteins (MAPs), which have been implicated in the regulation of microtubule assembly and function. MAP2 is the most abundant of these proteins, and it has been extensively characterized in various functional and pathological conditions. In the present study the distribution of MAP2 was examined in each layer of the CA1 and CA3 regions of the hippocampus proper and dentate gyrus in rat development. A total of 40 brains at various ages starting from postnatal day (P) 0 to P90 were examined. After perfusional fixation the brains were frozen and cut on the coronal plane and stained with either cresyl violet or standard immunohistochemical methods using the anti-MAP2 antibody. MAP2 exhibited a somatodendritic pattern of localization in cells of the hippocampus. Staining was most prominent in dendrites and perikarya as well as granules surrounding cell bodies. In a newborn rat's brain immunostaining was intense in granules and faint in perikarya. Between P4 and P21 immunostaining density for MAP2 was stronger and appeared in perikarya, granules, and dendritic trees. After P21 the perikarya and dendrites of the pyramidal layer and stratum radiatum of the hippocampus proper, as well as the molecular and granular layer of dentate gyrus, showed reduced immunoreactivity. In the stratum oriens of the hippocampus and polymorphic layer of the dentate gyrus immunoreactivity was still strong until P90.     

Developmental changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocampus

The laminar distribution of decreases in extracellular free calcium and concomitant field potentials induced by repetitive orthodromic stimulation, ionophoretic application of N-methyl-D-aspartate and quisqualate, was studied in the CA1 field of rat hippocampal slices, at two different stages during postnatal development. While stimulation-elicited and quisqualate-induced signals remain maximal in stratum pyramidale during the first postnatal month, the laminar profiles of responses to N-methyl-D-aspartate (NMDA) depend on age: the responses to this agonist are maximal in stratum pyramidale in 5-9-day-old rats and in stratum radiatum in 12-30-day-old rats. Our findings suggest that, during the second postnatal week, the apical dendrites of pyramidal neurons in area CAl become more sensitive to NMDA, which is expressed by big influxes of calcium at this level.     

Age-related changes in the rat hippocampus.

The human brain is uniquely powerful in its cognitive abilities, yet the hippocampal and neocortical circuits that mediate these complex functions are highly vulnerable during aging. In this study, we analyzed age-related changes in the rat hippocampus by studying newborn (1 month), middle-aged (12 months), and older (24 months) male and female Sprague-Dawley rats. We evaluated neuronal dystrophy, neuron scattering, and granulovacuolar degeneration in the hippocampal area using light microscopy, according to age and gender. We detected significant neuronal dystrophy in the CA1, CA2, and CA3 areas in male rats, and in the CA1, CA3, and CA4 areas in female rats. Degenerative changes, indicated by neuron scattering, were observed in the CA1, CA2, and CA3 areas of male and the CA2 and CA4 areas of female rats. Changes in all areas of the hippocampus were observed with increasing age; these changes included neuronal dystrophy and neuron scattering and did not differ significantly between male and female rats.     

Developmental changes in brain serotonin synthesis capacity in autistic and nonautistic children

Serotonin content, serotonin uptake sites, and serotonin receptor binding measured in animal studies are all higher in the developing brain, compared with adult values, and decline before puberty. Furthermore, a disruption of synaptic connectivity in sensory cortical regions can result from experimental increase or decrease of brain serotonin before puberty. The purpose of the present study was to determine whether brain serotonin synthesis capacity is higher in children than in adults and whether there are differences in serotonin synthesis capacity between autistic and nonautistic children. Serotonin synthesis capacity was measured in autistic and nonautistic children at different ages, using alpha[11C]methyl-L-tryptophan and positron emission tomography. Global brain values for serotonin synthesis capacity (K complex) were obtained for autistic children (n = 30), their nonautistic siblings (n = 8), and epileptic children without autism (n = 16). K-complex values were plotted according to age and fitted to linear and five-parameter functions, to determine developmental changes and differences in serotonin synthesis between groups. For nonautistic children, serotonin synthesis capacity was more than 200% of adult values until the age of 5 years and then declined toward adult values. Serotonin synthesis capacity values declined at an earlier age in girls than in boys. In autistic children, serotonin synthesis capacity increased gradually between the ages of 2 years and 15 years to values 1.5 times adult normal values and showed no sex difference. Significant differences were detected between the autistic and epileptic groups and between the autistic and sibling groups for the change with age in the serotonin synthesis capacity. These data suggest that humans undergo a period of high brain serotonin synthesis capacity during childhood, and that this developmental process is disrupted in autistic children.     

Developmental changes in serotonin levels in the chick spinal cord and brain

Developmental changes in 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the developing chick spinal cord and brain were examined using high-performance liquid chromatography with electrochemical detection and immunohistochemistry. On embryonic day (E)6 only small amounts of 5-HT (0.086 ng) and 5-HIAA (0.0144 ng) were found in the spinal cord. By contrast, large amounts of 5-HT (x30) and 5-HIAA (x60) were detected in non-neuronal tissue outside the spinal cord; a similar distribution of 5-HT was also detected by immunohistochemistry. Up to E10, the highest concentrations of 5-HT in the spinal cord were found in the cervical region, followed by the thoracic and lumbar regions. In embryos older than E16, as well as in posthatched chicks, however, the highest and lowest concentrations of 5-HT were found in the lumbar and thoracic spinal cord, respectively. The concentration of spinal cord 5-HT reached maximal values on posthatching day (P)7, after which there was a marked decrease. By P120, 5-HT levels in the spinal cord decreased to the same level as on E10-E16. Concentrations in the brain, however, gradually increased with development. The basic pattern of development of 5-HIAA was similar to that of 5-HT.     

Pre- and postsynaptic actions of adenosine in the in vitro rat hippocampus

The mechanisms underlying the depressant effect of adenosine on excitatory synaptic transmission were studied in rat hippocampus in vitro. The relative contribution of direct effects of adenosine upon CA1 pyramidal neurons (hyperpolarization, increased conductance) was evaluated by comparing the effects of superfused adenosine on EPSP amplitude, and on depolarizing responses to local application of glutamate. Adenosine depressed synaptic EPSPs to a greater extent than glutamate responses in 30 out of 32 cases, and its effects were independent of the site of glutamate application (somatic vs dendritic). Thus, the postsynaptic effects of adenosine, including a possible dendritic conductance that would be undetectable with somatic recordings, can only partially account for the depression of synaptic potentials observed with adenosine.     

Depolarization-dependent actions of dihydropyridines on synaptic transmission in the in vitro rat hippocampus

Field potential and intracellular recordings were obtained in the in vitro hippocampal slice to study the effects on synaptic transmission of dihydropyridine (DHP) derivatives. Nimodipine or nifedipine by itself had little effect upon the postsynaptic response as determined by field potential analysis. However, facilitation became evident when DHP application was coupled with manipulations which induced a moderate degree of membrane depolarization. In accordance with the hydrophobic nature of these compounds, extensive washing in normal Krebs' solution failed to reverse the facilitation indicating that the DHP effects outlasted the induced depolarization. Nifedipine is photolabile and its actions were reversed when intense light was applied to the slice. Application of the DHP Bay K 8644, resulted in a similar depolarization-dependent increase in neuronal excitability which, upon washout and exposure to light, was at first attenuated and then reversed, resulting in a long-lasting depression of the EPSP that was sensitive to caffeine. This depressant action of Bay K 8644 appeared to be mediated at a site presynaptic to the pyramidal cell because the postsynaptic component of the field potential response to pulsed applications of glutamate was not altered. Intracellular recording from CA1 neurons supports a presynaptic locus for the depressant actions of Bay K 8644; spike threshold for synaptically evoked responses was greatly increased while spike threshold to direct depolarization of the soma was unchanged. These results indicate that DHPs can exert effects on synaptic transmission in hippocampal brain slice under conditions of moderate membrane depolarization.     

Unraveling the modulatory actions of serotonin on male rat sexual responses

Animal studies and clinical investigations reveal that serotonin plays a central role in the control of the ejaculatory threshold. The chronic use of selective serotonin reuptake inhibitors (SSRIs) frequently results in sexual dysfunction, inviting to analyze the modulatory actions of serotonin on male sexual function in depth. Even though the main effect of serotonin on male sexual responses is inhibitory, this neuromodulator also mediates brief important stimulatory actions. Serotonin (5-HT) can activate two intracellular signaling pathways: a lower-threshold facilitatory pathway, and a higher-threshold inhibitory pathway, leading to biphasic effects. We propose that these divergent actions are related to the stimulation or inhibition of glutamatergic and GABAergic interneurons. Experimental evidence suggests that low 5-HT concentrations produce stimulatory actions on male ejaculatory aspects that might be mediated by the blockade of the GABAergic neurotransmission in the MPOA and spinal cord, which in turn releases a tonic inhibition that allows other neurotransmitters such as glutamate, noradrenaline, oxytocin and dopamine to initiate a sequence of molecular events resulting in the expression of ejaculation. Similar serotonin actions, mediated via interneurons, have been proposed for the regulation of other processes and occur in many central nervous system areas, indicating that it is not an isolated phenomenon.     

Pre- and postsynaptic actions of serotonin on rat suprachiasmatic nucleus neurons

Serotoninergic transmission is implicated in the photic and non-photic regulation of circadian rhythms. 5-HT (1-100 microM), carboxamidotryptamine (5-CT 0.1-10 microM) and (+)-8-hydroxy-dipropylaminotetraline (8-OH-DPAT, 1-30 microM) dose-dependently activated an outward current (5-100 pA) in 30% of neurons voltage-clamped at -60 mV in the suprachiasmatic nucleus (SCN) in vitro slice. EC(50) values were 7.0 microM for 5-HT and 0.2 microM for 5-CT. Serotonin-induced outward current was associated with an increase in input conductance, and the current was blocked by Ba(2+) (1 mM). The amplitude of the current was enhanced by depolarization, reduced by hyperpolarization, and reversed its polarity during a hyperpolarization beyond the potassium equilibrium potential. Mean amplitudes of the 5-HT outward current changed with time of the subjective circadian day. The value near CT2 (23.8 pA) was about 4 times greater than that around CT14 (6.7 pA). Cells that responded with an outward current showed four types of morphology: monopolar, simple bipolar, curly bipolar and radial shaped; they were localized in all parts of the SCN. The EPSC evoked by retino-hypothalamic-tract (RHT) stimulation was inhibited 26% but the inward current induced by exogenously applied glutamate or NMDA was not affected by serotonin agonists. Focal stimulation-induced and spontaneous IPSC but not the exogenous GABA-induced outward current were inhibited by 5-HT agonists in a subpopulation of cells. In conclusion, 5-HT regulates SCN neurons by both pre- and post-synaptic inhibitory mechanisms; the latter may play a key role in modulating SCN circadian rhythm by activation of 5-HT receptors and opening of a potassium channel.     

Cytoskeletal changes in the hippocampus following restraint stress: role of serotonin and microtubules

The aetiology of depression is associated with depletion in central levels of serotonin (5-HT). Hence, a major effect of antidepressant drugs is to increase synaptic 5-HT levels. Stressful conditions have also been shown to affect neuronal plasticity and 5-HT neurotransmission in the hippocampus. Neuronal plasticity, which is typically referred to as a structural adaptation of neurons to functional requirements, requires more dynamic forms of microtubules (cytoskeletal component). The alpha-tubulin, which is the major component of microtubules, can be postranslationally modified and both the tyrosinated (tyr-tub) and acetylated (acet-tub) forms are considered markers of more dynamic or more stable microtubules, respectively. The aim of the present work was to investigate the expression of tyr-tub and acet-tub in the hippocampus of rats submitted to either acute (6 h for 1 day) or sub-chronic (6 h for 4 days every day) restraint stress. In addition, ex vivo hippocampal 5-HT levels were monitored by differential pulse voltammetry to analyse the influence of both stress conditions upon 5-HT levels. Our results showed that the expression of tyr-tub in the hippocampus was significantly decreased to 70 +/- 7% following sub-chronic restraint stress (P < 0.01). In contrast, acute and sub-chronic restraint stress increased the hippocampal expression of acet-tub to 139 +/- 11% and 145 +/- 11% of control, respectively. Finally, 5-HT levels were significantly increased (P < 0.05) to 142 +/- 15% and 135 +/- 11% following acute and sub-chronic restraint stress, respectively. The stress-induced cytoskeletal changes observed in the present study suggest that the microtubular network is a potential new pathway that may increase our understanding of stress-related events.     

Developmental changes in the number,size, and orientation of GFAP-positive cells in the CA1 region of rat hippocampus

Changes in extracellular potassium concentration as measured with ion-selective microelectrodes revealed abnormally large accumulations in the hippocampus during postnatal development. While rises in [K⁺]_o during stimulation of the Schaffer collaterals were limited to about 12 mM in adult animals, identical stimulations elicited rises to levels as large as 18 mM in juveniles. Since astrocytes are believed to play an important role in K⁺ homeostasis, we studied the postnatal development of astrocytes in the CA1 region of rat hippocampus in four age groups using a polyclonal antibody against glial fibrillary acidic protein (GFAP). The main proliferation of GFAP-positive cells (GFAPpc) occurred in all laminae between postnatal days 8 and 16. The number of GFAP-positive astrocytes per unit area was reached in stratum lacunosum-moleculare and stratum oriens at about 2 weeks and in stratum radiatum at about 3 weeks of age. During further development—at the age of 24 days—the orientation of individual astrocytes in stratum radiatum became polar with an orientation almost perpendicular to stratum pyramidale. This was revealed by an analysis based on determination of the quotients between the angular orientation of the processes of single individual GFAP-positive cells. When the crossing points of all glial processes over vertical and horizontal grid lines were determined and respective quotients evaluated, the same development towards a perpendicular orientation of astrocytes was noted in stratum radiatum. The same approach revealed a transient orientation parallel to the fissure in stratum lacunosum-moleculare around day 24. Camera lucida drawings of GFAPpc in stratum radiatum revealed that astrocytes became larger during the first three postnatal weeks, followed by a reduction of various parameters (e.g., cell extension, branching pattern) until adulthood. The observed developmental changes of astroglial cells may contribute to the known delayed maturation of potassium regulation in rat hippocampus.     

Developmental changes in mu suppression to observed and executed actions in autism spectrum disorders

There has been debate over whether disruptions in the mirror neuron system (MNS) play a key role in the core social deficits observed in autism spectrum disorders (ASD). EEG mu suppression during the observation of biological actions is believed to reflect MNS functioning, but understanding of the developmental progression of the MNS and EEG mu rhythm in both typical and atypical development is lacking. To provide a more thorough and direct exploration of the development of mu suppression in individuals with ASD, a sample of 66 individuals with ASD and 51 typically developing individuals of 6–17 years old were pooled from four previously published studies employing similar EEG methodology. We found a significant correlation between age and mu suppression in response to the observation of actions, both for individuals with ASD and typical individuals. This relationship was not seen during the execution of actions. Additionally, the strength of the correlation during the observation of actions did not significantly differ between groups. The results provide evidence against the argument that mirror neuron dysfunction improves with age in individuals with ASD and suggest, instead, that a diagnosis-independent developmental change may be at the root of the correlation of age and mu suppression.     

Developmental changes in microglial mobilization are independent of apoptosis in the neonatal mouse hippocampus

During CNS development, microglia transform from highly mobile amoeboid‐like cells to primitive ramified forms and, finally, to highly branched but relatively stationary cells in maturity. The factors that control developmental changes in microglia are largely unknown. Because microglia detect and clear apoptotic cells, developmental changes in microglia may be controlled by neuronal apoptosis. Here, we assessed the extent to which microglial cell density, morphology, motility, and migration are regulated by developmental apoptosis, focusing on the first postnatal week in the mouse hippocampus when the density of apoptotic bodies peaks at postnatal day 4 and declines sharply thereafter. Analysis of microglial form and distribution in situ over the first postnatal week showed that, although there was little change in the number of primary microglial branches, microglial cell density increased significantly, and microglia were often seen near or engulfing apoptotic bodies. Time-lapse imaging in hippocampal slices harvested at different times over the first postnatal week showed differences in microglial motility and migration that correlated with the density of apoptotic bodies. The extent to which these changes in microglia are driven by developmental neuronal apoptosis was assessed in tissues from BAX null mice lacking apoptosis. We found that apoptosis can lead to local microglial accumulation near apoptotic neurons in the pyramidal cell body layer but, unexpectedly, loss of apoptosis did not alter overall microglial cell density in vivo or microglial motility and migration in ex vivo tissue slices. These results demonstrate that developmental changes in microglial form, distribution, motility, and migration occur essentially normally in the absence of developmental apoptosis, indicating that factors other than neuronal apoptosis regulate these features of microglial development.     

Cholinergic denervation-like changes in rat hippocampus following developmental lead exposure

We investigated the effects of developmental lead exposure from embryonic day 16 (E16) through postnatal day 28 (PN28), on cholinergic and catecholaminergic markers in the septohippocampal pathway in rats through fourth month of age. Lead exposure resulted in a persistent 30–40% reduction of [³H]hemicholinium-3 ([³H]HC-3) binding in the hippocampus through PN120, and 20–30% reduction of septal and hippocampul choline acetyltransferase (ChAT) activity which persisted through PN84 but returned to control levels in both septum and hippocampus at PN112. The muscarinic ligand [³H]quinuclidinyl benzylate ([³H]QNB) binding was reduced in the septum at PN28 but did not differ significantly from controls at PN56–PN112. Neither short- nor long-term effects of Pb exposure on [³H]QNB binding were seen in the hippocampus. Similar to the effects of fimbria-fornix transection, Pb exposure resulted in a long-term 50–90% increase of tyrosine hydroxylase(TH) activity in the hippocampus, although neither treatment affected TH activity in the septum. The lead-induced increase in hippocampul TH was significantly attenuated by superior cervical ganglionectomy. It is concluded that the effects of perinatal lead exposure resemble in several respects those seen following surgical disruption of the septohippocampal pathway in adult animals. The denervation-like effects in the hippocampus may be an important factor in long-term learning and cognitive impairments following developmental exposure to low-levels of lead.     

In vivo microdialysis measures of extracellular serotonin in the rat hippocampus during sleep-wakefulness.

We investigated extracellular 5-hydroxytryptamine (5-HT) levels in rat hippocampus during different stages of the sleep-waking cycle using in vivo microdialysis. The extracellular 5-HT level was highest in active waking (AW) and, when compared to AW, 5-HT level was progressively lower in quiet waking (QW; 78%), quiet sleep (QS; 50%) and REM (which we termed active sleep (AS); 40%). Functional implications of AS related-decreased 5-HT in the hippocampus are discussed.     

Developmental changes in purine nucleotide metabolism in cultured rat astroglia

The present study was conducted in order to clarify the role of the glia in brain purine metabolism. This, in connection with the clarification of the etiology of the neurological manifestations associated with some of the inborn errors of purine metabolism in man. Purine nucleotide content, the capacity for de novo and salvage purine synthesis and the activity of several enzymes of purine nucleotide degradation, were assayed in primary cultures of rat astroglia in relation to culture age. The capacity of the intact cells to produce purine nucleotides de novo exhibited a marked decrease with the culture age, but the activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), catalyzing salvage nucleotide synthesis, increased. Aging was also associated with a marked increase in the activity of the degradation enzymes AMP deaminase, purine nucleoside phosphorylase (PNP) and guanine deaminase (guanase). The activity of adenosine deaminase and of AMP-5′-nucleotidase, increased markedly during the first 17 days in culture, but decreased thereafter.

The results indicate that purine nucleotide metabolism in the cultured astroglia is changing with aging to allow the cells to maintain their nucleotide pool by reutilization of preformed hypoxanthine, rather than by de-novo production of new purines. Aging is also associated with increased capacity for operation of the adenine nucleotide cycle, contributing to the homeostasis of adenine nucleotides and to the energy charge of the cells. In principle, the age-related alterations in purine metabolism in the astroglia resemble those occurring in the maturating neurons, except for the capacity to produce purines de novo, which exhibited inverse trends in the two tissues. However, in comparison to the neurons, the cultured astroglia possess the capacity for a more intensive metabolism of purine nucleotides.