Immature Neocortex Research Articles

Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones.

1. Developmental changes in electrophysiological properties of pyramidal neurones correlated with the developmental decline in gap junction-dependent dye coupling were investigated in coronal slices of rat prefrontal and sensorimotor cortex. Effects of intracellular acidification induced by application of weak organic acids on neuronal dye coupling, electrotonic parameters as well as synaptic potentials were examined using the patch clamp technique. Optical monitoring of intracellular pH revealed an acidic shift of 0.4-0.5 pH units following sodium propionate application. 2. Dye coupling between layer II-III neurones was prominent during the first two postnatal weeks. During this period, pre-incubation of slices with 30 mM of the sodium salts of weak organic acids reduced the number of cells coupled to the injected neurones by 64%. 3. Between postnatal days 1 and 18, the mean neuronal input resistance decreased significantly (by 81.0%). Both the membrane time constant (tau 0) and the first equalizing time constant (tau 1) also showed a significant developmental decline of 25.8 and 65.8%, respectively. Electrotonic length decreased by 34.9%. The electrophysiological properties of neurones displayed a pronounced intercellular variability which decreased with on-going development. 4. During the first two postnatal weeks, intracellular acidification led to a mean increase in neuronal input resistance of 55.9% and a mean decreae in electrotonic length of 22.2%. The membrane time constant was reduced by approximately 25% in the majority of neurones tested. Significant electrophysiological effects induced by intracellular acidification were not detected in uncoupled neurones from 18-day-old rats. 5. EPSP width at half-maximal amplitude showed a substantial reduction of approximately 50%, while rise times of the non-NMDA receptor-mediated EPSP components displayed no significant change during development. Both weak organic acids, as well as the gap junction blocker 1-octanol, reduced excitatory synaptic transmission independent of developmental age. 6. We conclude that gap junction permeability is regulated by intracellular pH in developing layer II-III pyramidal cells in the rat neocortex. The prominent correlation between pH-induced reduction in dye coupling and changes in electrophysiological cell properties suggests a significant influence of gap junctions on synaptic integration and information transfer in the immature neocortex.

Modulation of epileptiform activity by metabotropic glutamate receptors in immature rat neocortex.

1. Intracellular and extracellular recordings were obtained from neocortical brain slices of immature rats (postnatal days 9-16) maintained in vitro. Spontaneous and evoked epileptiform discharges (termed paroxysmal depolarizing shifts or PDSs) were recorded from upper cortical laminae (layers II-III) after exposure to the gamma-aminobuturic acid-A receptor antagonist, bicuculline methiodide. The effects of mGluR activation on PDS duration, spontaneous frequency, and threshold for evoking a PDS were determined. Putative mGluR agonists and antagonists also were tested. 2. Bath application of the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD, 50-200 mM) elicited biphasic, time-dependent effects on evoked and spontaneous epileptiform discharges. At times early in drug wash-in, ACPD increased PDS duration and spontaneous PDS frequency. In > 60% of the slices, the spontaneous PDSs became regular. Subsequently, ACPD reduced PDS duration and increased the stimulus threshold for evoking a PDS, suggesting that the actions of ACPD were dose dependent. 3. Investigation of the concentration-dependence revealed that sustained low ACPD concentrations (5 microM) elicited only facilitatory actions, whereas higher concentrations were suppressive. These observations suggest the activation of different mGluR subtypes, which may be localized differentially at pre- and postsynaptic sites. 4. Bath application of the mGluR agonists, L-2-amino-4-phosphonobutyrate or (2S,3S,4S)-alpha-(carboxycyclopropyl) glycine, produced only suppressive effects on epileptiform activity in the immature neocortex. L-2-amino-3-phosphonopropionate was an ineffective antagonist of ACPD-mediated modulation of epileptiform activity. Application of the putative antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG), failed to antagonize the biphasic actions of ACPD. MCPG had suppressive effects of epileptiform activity, suggesting activation of mGluRs by endogenous agonists. 5. Simultaneous recordings from deeper and upper cortical layers indicated that the initial negativity of both evoked and spontaneous PDSs began in deeper cortical layers under control conditions and in the presence of ACPD. Intracellular records from neurons in deeper layers displayed two distinct patterns of activity during mGluR activation. Most deep layer neurons received a barrage of excitatory postsynaptic potentials before a spontaneous PDS during ACPD application. A small population of neurons depolarized and entered a tonically firing mode, which was interrupted by spontaneous PDSs. Different neuronal populations, possible expressing different mGluR subtypes or coupling mechanisms, may play integral roles in the induction and generation of epileptiform activities. 6. Thapsigargin or dantrolene, agents thought to block release of Ca2+ from intracellular stores, were both applied for periods < or = min.(ABSTRACT TRUNCATED AT 400 WORDS)

Reduced Mg2+ block of N-methyl-D-aspartate receptor-mediated synaptic potentials in developing visual cortex.

Molecular cloning has demonstrated a diversity of artificially expressed N-methyl-D-aspartate (NMDA) receptors, implying a similar diversity of naturally occurring NMDA receptors in situ. Particularly significant was the success in expression of NMDA receptor classes exhibiting various sensitivities to Mg2+ block, a voltage-dependent channel blockade by Mg2+ that is essential to NMDA receptor functioning. Release from Mg2+ block often allows or facilitates the occurrence of long-term potentiation, a form of synaptic plasticity. Here we show that in the immature visual cortex, which is more susceptible to long-term potentiation than adult visual cortex, synaptically activated NMDA receptors, unlike those in the adult, have varying but clearly reduced sensitivities to Mg2+ block. We propose that the initially expressed, later-eliminated NMDA receptors exhibiting a reduced Mg2+ block may underlie the greater susceptibility to plasticity in the immature neocortex.

Synthesis of the foetal protein fetuin by early developing neurons in the immature neocortex.

The presence of the foetal protein fetuin has previously been demonstrated by immunocytochemistry to be specifically confined to the primordial plexiform layer, the early cortical plate and subplate zone cells in the developing neocortex of a number of species. In order to investigate its origin there, we have applied in situ hybridization in paraffin sections of Bouin's fixed foetal sheep brain, using a short anti-sense oligonucleotide probe. The distribution of fetuin mRNA has been compared with that of the protein by using anti-fetuin antibodies and immunocytochemistry. This allowed us to confirm that fetuin is synthesised initially in cells of the primordial plexiform layer and subsequently cortical plate and subplate cells. On the other hand, cells in the ventricular zone that are fetuin (protein) positive do not contain detectable fetuin mRNA. The time course of fetuin mRNA expression in the developing neocortex follows closely the previously described pattern of fetuin (protein) distribution in the sheep brain, apart from its absence from the ventricular zone where its origin is probably by uptake from cerebrospinal fluid.

NMDA receptor involvement in epileptogenesis in the immature neocortex.

NMDA receptor involvement in epileptogenesis in the immature neocortex.

Excitatory synaptic involvement in epileptiform bursting in the immature rat neocortex

1. Neocortical brain slices were prepared from animals 8-15 days of age and maintained in vitro. Intracellular recordings were obtained from neurons in cortical layers 2-3. The role of synaptic activity and excitatory amino acid receptors in generation of picrotoxin-induced ictal-like epileptiform activity in the immature neocortex was investigated. D-2-amino-5-phosphonovaleric acid (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were used as selective antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. 2. Ictal-like epileptiform discharges were induced by bath application of the GABAA-receptor antagonist picrotoxin. Paroxysmal discharges, 7-25 s in duration, occurred spontaneously or could be evoked by electrical stimulation. These events consisted of an initial paroxysmal depolarizing shift (PDS) followed by a long-duration depolarization (LLD) with superimposed late PDSs. 3. The amplitudes of the initial PDS, LLD, and late PDSs were linearly dependent on membrane potential, increasing with hyperpolarization and diminishing on depolarization. All responses reversed polarity near 0 mV. Under voltage-clamp conditions, both transient and sustained currents were observed, coincident with PDSs and the LLD, respectively. The duration of the ictal-like events was similar under current- and voltage-clamp conditions, suggesting activation of intrinsic membrane currents did not significantly prolong epileptiform discharges. 4. Bath application of D-APV (20 microM) decreased the amplitude and duration of both the initial PDS and LLD without affecting the time-to-onset of epileptiform activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Monodelphis domestica (grey short-tailed opossum): an accessible model for studies of early neocortical development

The development of the neocortex of the marsupial Monodelphis domestica has been studied from birth until adulthood. Monodelphis is born after a gestational period of 14 days, a time when the neocortex is still at a two-layered "embryonic" stage of development, that is equivalent to a 13-14 day rat embryo or 6 week human embryo. The cortical plate does not begin to appear until 3 to 5 days postnatal. Thus the whole of neocortical development is a postnatal phenomenon in this species, as has been previously described in other marsupials. The general pattern of development of the characteristic layers of the immature neocortex and the subsequent development of a six-layered adult neocortex is similar to that found in eutherian species. However there are some differences. The depth of the immature cortical plate when compared to the thickness of the neocortical wall is less than in eutherians and the subplate zone is much deeper in Monodelphis; this transient subplate zone consists of widely spaced rows of cells that are aligned parallel to the cortical surface. Unlike eutherians there appears to be no secondary proliferative zone in the subventricular zone of the dorso-lateral neocortical wall. Maturation of the neocortex is apparent by 45 days postnatal and by 60 days (around the time of weaning) the characteristic six-layered adult neocortex is clearly present. The neuronal marker PGP 9.5 was used to define neuronal populations in the adult brain. The density of neurons in Monodelphis appears to be considerably less than in eutherians such as the rat. The suitability of postnatal Monodelphis for studies of neocortical development is discussed.

Alterations in the microenvironment during spreading depression associated with epileptiform activity in the immature neocortex

Local changes in extracellular ion concentrations were measured with ion-sensitive microelectrodes in slices of mature (>40 days of age) or immature (16–30 days of age) rat neocortex maintained in vitro. Repetitive stimulation resulted in increases in extracellular potassium ([K +] o) to levels of 8.85 ± 2.1 mM in slices from adult animals and 12.77 ± 1.8 mM in slices from immature animals. During exposure to picrotoxin, maximum levels were 11.3 ± 2.6 and 14.8 ± 2.5 mM in the mature and immature groups, respectively. Picrotoxin (50 μM) induced spontaneous bursts of repetitive spiking, followed by a slow, negative field potential, associated with spreading depression (SD), in slices from immature animals. [K +] o levels increased to 10.2 ± 3.9 mM during repetitive spike discharges and reached 30.3 ± 18.5 mM during SDs. Variations in the size of the extracellular space (ES) were examined during SD. The ES was found to reversibly decrease by 39 ± 4.5%. Clusters of repetitive spikes were associated with 0.1–0.2 mM decreases in [Ca 2+] o, whereas 1.12 ± 0.06 mM decreases were observed during SDs. Decreases in [Na +] o and [Cl - o of 56 ± 10 mM and 41 ± 9 mM, respectively, were observed during SDs suggesting that a net transmembrane movement of water occurred during SDs. These results indicate that changes in [K +] o associated with epileptiform activity in the immature nervous system are quantitatively different from those observed in the mature brain. These large increases in [K +] o may contribute to the prolonged nature of epileptiform discharges in the developing nervous system.

Zinc and glycine do not modify low-magnesium-induced epileptiform activity in the immature neocortex in vitro

Exposure of neocortical slices from immature rats to saline containing no added magnesium induced spontaneous epileptiform activity that consisted of bursts of low-amplitude isolated discharges lasting 50–90 sec, recurring every 90–300 sec. Bath application of the N-methyl- d-aspartate (NMDA) receptor antagonist dl-2-amino-7-phosphonoheptanoic acid led to a rapid, reversible suppression of epileptiform activity, indicating involvement of NMDA receptors. Perfusion with zinc or glycine, putative modulators of the NMDA receptor, with suppressive and enhancing properties, respectively, had no effect on the frequency or duration of the epileptiform discharges. These results indicate that in the immature neocortex in vitro, application of zinc or glycine does not modulate NMDA receptor-mediated, low-magnesium-induced epileptiform discharges.

Extracellular K + and Ca 2+ changes during epileptiform discharges in the immature rat neocortex

Picrotoxin-induced epileptiform activity was examined in neocortical slices prepared from 8- to 15-day-old rats. This activity consisted of spontaneous bursts of 3–5 discharges that resembled interictal spikes and were interspersed with ictal-like paroxysms lasting 10–30 s. Measurements of extracellular potassium ([K +] o) and calcium ([Ca 2+] o) were made during these spontaneous epileptiform events, using ion-sensitive electrodes. Individual interictal spikes were associated with [Ca 2+] o decreases of 0.1–0.2 mM, whereas sustained ictal-like discharges were accompanied by decreases of 0.3–0.4 mM. Measurement of [K +] o showed that individual interictal spikes were associated with increases in [K +] o up to 12 mM, whereas increases to more than 20 mM accompanied long-lasting ictal-like discharges. Maximum increases in [K +] o were observed ca. 600 μm below the pial surface. [K +] o increases were followed by undershoots of the resting [K +] o level. The unusually high [K +] o levels associated with epileptiform discharges in the immature neocortex suggests that disturbances in [K +] o regulation may contribute to the generation of the picrotoxin-induced, spontaneous, prolonged ictal-like discharges observed in the 8- to 15-day age group.

Muscarinic cholinergic receptors in human infant forebrain: [ 3H]Quinuclidinyl benzilate binding in homogenates and quantitative autoradiography in sections

The ontogeny of muscarinic receptors in human brain was studied by comparing [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding in postmortem tissue from infants 1 week to 3 months of age with binding in adult specimens. Saturation analysis with [ 3H]QNB and displacement studies with muscarinic antagonists and agonists in tissue homogenates demonstrated that binding sites in the infants' forebrain regions were present in adult or higher than adult concentrations (B max). Binding affinity ( K d ) and pharmacological characteristics were nearly identical at the two ages. Quantitative receptor autoradiography demonstrated more [ 3H]QNB binding in the gray matter of infants than adults and revealed a marked difference between the two ages in the laminar distribution of binding sites in neocortex. In contrast to the adult pattern with higher binding in superficial layers 1–3 than in layers 4–6, the distribution in the immature cortex was inverted. These results suggest that muscarinic receptors in infants resemble closely those in mature brain. However, the topography of receptors in the immature neocortex is distinct and they are redistributed in a gradient from inside outward during postnatal development.

Characterization of monoaminergic terminals in the neostriatum of neonatal rats: an electron microscopic morphometric analysis.

Monoaminergic (MA) boutons in the neostriatum of neonatal rats were studied by using 5-hydroxydopamine. We found: (a) bouton size averaged 0.6 micron in diameter; (b) terminal density was 68 boutons per 7200 micron2 area; (c) synaptic frequency was 33.2%; and (d) MA neurons constituted 9.2% of all boutons. This population was much lower than that reported in immature neocortex where MA neurons are a major innervation.

Horseradish peroxidase pellets implanted into infant neocortex: Some technical considerations

This study was undertaken to examine whether implanting pellets of horseradish peroxidase (HRP), rather than injecting an aqueous solution, would improve the sensitivity of the retrograde tracing method as applied to infant rat neocortex. From 1 to 10 pellets, each containing approximately 10 μg of HRP, were implanted into somatosensory cortex of 6-day-old rats. Implantation of one pellet labeled 4 neuronal groups; 5 pellets, 37 groups. Higher doses of injected HRP (20–50 μg and 200–400 μg) are needed to label the same number of groups. Also, individual neurons of a group generally contain more granules/cell after pellets than following injections of much higher doses of HRP. The pellet implant technique offers a high degree of reproducibility and is technically simpler than injections. We conclude that HRP pellet implants offer advantages over injections in identifying potential afferents to immature neocortex.

Development of neocortical circuitry: Quantitative ultrastructural analysis of putative monoaminergic synapses

The distribution, numbers and morphology of presumed monoaminergic (MA) synapses were examined in somatosensory cortex of neonatal rats and mice (newborn to 16 days of age). MA synapses were identified using an ultrastructural cytochemical marker, 5-hydroxydopamine (5-OHDA), which results in the appearance of small granular vesicles (SGV) in their presynaptic terminals. From birth to 7 days of age, 20–30% of all synapses sampled in somatosensory cortex contain SGVs. However, few SGV synapses are seen in 8-day-old cortex and by 12 days of age, SGVs are no longer detectable in cortex. A specific laminar distribution for these SGV synapses—which is distinct from the overall synaptic distribution—is first seen at 3 days of age and is essentially unchanged until 7 days postnatally. During this entire period, the SGV synapses predominate in the primordium of layer IV, where they account for 50–70% of all synapses. Morphometric analysis of SGV synapses indicates that there are differences in junctional symmetry, vesicle shape and configuration of the contact zone between SGV and non-SGV synapses, as well as between SGV synapses themselves in the various cortical layers. The laminar distribution and morphological characteristics of SGV synapses suggest that the MA projection to neocortex exhibits a high degree of spatial specificity during its ingrowth. Also, the relatively high proportion of SGV synapses in the first postnatal week may reflect a potent influence exerted by the MA inputs on immature neocortex. The decreased numerical density of SGV synapses after 7 days of age is probably due to the development of the blood-brain barrier to 5-OHDA.

Acetylcholinesterase-containing neurons of layer VIb in immature neocortex: possible component of an early formed intrinsic cortical circuit.

In this ontogenetic study the neurons of layer VIb of rodent somatosensory cortex have been characterized using acetylcholinesterase (AchE) histochemistry, Golgi, and electron microscopic techniques. Already, at birth, the neurons were found to be AchE-rich. They contain presumed AchE reaction product within their granular endoplasmic reticulum. These cells send fine axons upwards towards the subpial layer where they terminate in a dense, AchE-rich, fiber plexus. In chronically undercut cortex, AchE staining persists in layer VIb neurons and in the subpial fiber plexus. These observations continue to support the view that there is an intrinsic neuronal circuit connecting layer VI with I in immature neocortex. The findings also raise the possibility that the putative circuit is cholinergic.

Modification of Ontogenetic Patterns in Mammalian Brain

Publisher Summary Normal morphological and physiological developmental patterns in feline cerebral and cerebellar cortex are summarized in this chapter in order to provide a basis for the analysis of the pathophysiological effects of two types of injuries to the immature brain. Surgical interruption of axons of pyramidal neurons markedly accelerates the development of intracortical axon-collaterals that establish synaptic relations with adjacent arciform pyramidal neurons. One consequence of this re-organization of synaptic pathways is reflected in the development of powerful excitatory synaptic activities in traumatized regions of immature neocortex. Effects of x-irradiation of cerebellar cortex in newborn kittens are defined primarily in terms of loss of the external granular layer and modification of Purkinje cell morphogenesis. Major alterations in electrophysiological properties of irradiated cerebellar cortex are seen in evoked responses to folial surface and motor cortex stimulation. These alterations are considered expressions of radiation-induced maturation acceleration and re-organization of synaptic pathways and possible changes in membrane properties of Purkinje cell dendrites. The data obtained in these ontogenetic studies of pathophysiological processes indicate that different types of insults to the immature brain may produce similar overt effects, that is, changes in excitability by entirely different mechanisms.

Properties of synaptic activities and spike potentials of neurons in immature neocortex.

Properties of synaptic activities and spike potentials of neurons in immature neocortex.

Morphological and physiological properties of chronically isolated immature neocortex

Areas of neocortex were neuronally isolated in neonatal kittens and studied 2 to 9 days later. Electrocortical activities in these preparations were compared with evoked responses of acutely isolated neocortex in kittens 6 to 10 days old. Characteristics of superficial (negative) cortical responses in both acute and chronic preparations were similar to those previously reported for intact immature neocortex. Synaptic activation of elements in the cortical depths was readily elicited by weak surface stimulation of chronically isolated slabs, but was rarely observed in acute preparations. Complex 8 to 14 per second repetitive bursts were evoked only in chronic preparations. These bursts were restricted in distribution and remarkably stable in overt configuration. Predominantly surface-positive evoked repetitive responses were associated with subsurface focal negativities of maximum amplitude in regions corresponding to the lower limits of the isolated slab. Low-threshold sites for initiating repetitive bursts were found at similar locations in the cortical depths. Topically applied GABA first augmented early surface-positive components of the repetitive sequences, then eliminated them. Convulsant agents that block inhibitory postsynaptic potentials (ϵ-aminocaproic acid, C 6, and strychnine) also depressed evoked repetitive bursts, but the depression produced by C 6 was preceded by a phase of augmented surface and subsurface negativity. Attempts were made to correlate these observations with histological (Golgi-Cox) studies of chronically isolated immature neocortex. The postnatal development of cortical neurons was found to be unaffected by the isolation procedure carried cut in the immediate neonatal period. Pyramidal neurons, the main stem axons of which had been severed during the isolation procedure, possessed numerous axon collaterals with extensive intracortical ramifications. This conversion of Type I pyramidal neurons into Type II arciform cells of Cajal in chronically isolated immature cortex is considered to be the major factor responsible for the increase in excitatory synaptic drives that are reflected in evoked repetitive bursts.