Cellular physiology of the neonatal rat cerebral cortex

The early development of the cerebral cortex is characterized by neurogenesis, neuronal migration, cellular differentiation and programmed cell death. Cajal-Retzius cells, developing cortical plate neurons and subplate cells form a transient synaptic circuit which may serve as a template for the formation of cortical layers and columns. These three neuronal cell types show distinct electrophysiological properties and synaptic inputs. Endogenous or exogenous harmful disturbances during this developmental period may lead to the preservation of early cortical circuits, which may act as trigger zones for the initiation of pathophysiological activity.     

Cellular morphology and physiology of the perinatal rat cerebral cortex

The cellular morphology and electrophysiology of the rat neocortex between embryonic day (E) 18 and postnatal day (P) 3 was studied in vitro by extracellular biocytin injections and whole-cell recordings, respectively. Most neurons were characterized by a small number of short-range dendrites and a main axon that was directed towards the white matter. Biocytin injections into the marginal zone and the cortical plate labeled far-reaching connections extending up to 2 mm in horizontal direction, indicating the existence of a dense network of long-range intrinsic projections in the neonatal cortex. Action potentials could be elicited as early as E18 and repetitive firing could first be observed at P0. Electrical stimulation of the immature cortex at various positions elicited polyphasic and long-lasting (up to 1 s) excitatory postsynaptic potentials and currents, which were significantly reduced in amplitude by a selective N-methyl-D-aspartate receptor antagonist. Our data indicate that the perinatal cortex manifests the structural and functional conditions for powerful excitatory interactions, which increase the likelihood for the generation of epileptiform activity during this developmental period. Copyright Copyright 1999 S. Karger AG, Basel     

Spontaneous GABAergic postsynaptic currents in Cajal-Retzius cells in neonatal rat cerebral cortex

Cajal-Retzius cells are among the first neurons appearing during corticogenesis, and play an important role in the establishment of cortical lamination. The variety of neurotransmitter receptors recently found on these cells imply that they are integrated in the neonatal cortical network. To investigate the presence and properties of spontaneous synaptic activity we performed whole-cell patch-clamp recordings from visually identified and biocytin-labelled Cajal-Retzius cells in a tangential slice preparation of neonatal rat cerebral cortex (postnatal days P0-P5). Spontaneous postsynaptic currents (sPSCs) could be observed in about 23% of the cells using a pipette solution containing 136 m M Cl-. The sPSCs occurred at a low frequency (0.07 +/- 0.07 Hz, n = 42 cells), had an average amplitude of 24.3 +/- 12.4 pA (n = 415 events) and could not be divided in subpopulations according to their amplitude distribution or kinetic properties. The sPSCs were blocked by the GABAA antagonist bicuculline (100 microM), while the glutamatergic antagonists (+/-)-2-amino-5-phosphonopentatonic acid (APV, 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), as well as tetrodotoxin (1-2 microM), a blocker of voltage-gated sodium-currents, had no significant effect on sPSCs. The incidence rate of sPSCs declined within the age of the rats and no sPSCs could be observed after P4. These results suggest that Cajal-Retzius cells transiently receive action potential-independent and GABA(A) receptor-mediated spontaneous synaptic input, which may contribute to the refinement of cortical circuits.     

Entorhinal cortex of the rat: Organization of intrinsic connections

Two sets of experiments were carried out to examine the organization of associational connections within the rat entorhinal cortex. First, a comprehensive analysis of the areal and laminar distribution of intrinsic projections was performed by using the anterograde tracers Phaseolus vulgaris–leuocoagglutinin (PHA-L) and biotinylated dextran amine (BDA). Second, retrograde tracers were injected into the dentate gyrus and PHA-L and BDA were injected into the entorhinal cortex to determine the extent to which entorhinal neurons that project to different septotemporal levels of the dentate gyrus are linked by intrinsic connections. The regional distribution of intrinsic projections within the entorhinal cortex was related to the location of the cells of origin along the mediolateral axis of the entorhinal cortex. Cells located in the lateral regions of the entorhinal cortex gave rise to intrinsic connections that largely remained within the lateral reaches of the entorhinal cortex, i.e., within the rostrocaudally situated entorhinal band of cells that projected to septal levels of the dentate gyrus. Cells located in the medial regions of the entorhinal cortex gave rise to intrinsic projections confined to the medial portion of the entorhinal cortex. Injections made into mid-mediolateral regions of the entorhinal cortex mainly gave rise to projections to mid-mediolateral levels, although some fibers did enter either lateral or medial portions of the entorhinal cortex. These patterns were the same regardless of whether the projections originated from the superficial (II–III) or deep (V–VI) layers of the entorhinal cortex. This organizational scheme indicates, and our combined retrograde/anterograde labeling studies confirmed, that laterally situated entorhinal neurons that project to septal levels of the dentate gyrus are not in direct communication with neurons projecting to the temporal portions of the dentate gyrus. These results suggest that entorhinal intrinsic connections allow for both integration (within a band) and segregation (across bands) of entorhinal cortical information processing. J. Comp. Neurol. 398:49–82, 1998. © 1998 Wiley-Liss, Inc.     

Glial responses to neonatal hypoxic-ischemic injury in the rat cerebral cortex.

Neurogenesis is nearly completed after birth, whereas gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. Little is known about the effects of hypoxic-ischemic (HI) injury on these critical postnatal processes. Here we explored the glial reactions to mild HI injury of the neonatal rat cerebral cortex at P3. We show that the HI lesion results in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. The morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggest an accelerated transformation of radial glia into astrocytes. In addition, BrdU/GFAP immunostaining revealed a significant increase of double-labelled cells indicating an acute proliferation of astrocytes after HI. This enhanced proliferative activity of astrocytes persisted for several weeks. We found an elevated number and increased mitotic activity of both NG2-positive oligodendrocyte progenitors and RIP-positive oligodendrocytes after injury. These findings imply that glial responses are central to cortical tissue remodelling following neonatal ischemia and represent a potential target for therapeutic approaches.     

Electrophysiological properties of neuroendocrine cells of the intact rat pars intermedia: multiple calcium currents

Intracellular recordings for current and voltage clamping were obtained from 130 neuroendocrine cells of the pars intermedia (PI) in intact pituitaries maintained in vitro. Spontaneous and evoked action potentials were blocked by TTX or by intracellular injection of a local anesthetic, QX-222. After potassium (K+) currents were blocked by tetraethylammonium (TEA), 4-aminopyridine, and intracellular cesium (Cs+), 2 distinct calcium (Ca2+) spikes were observed which were differentiated by characteristic thresholds, durations, and amplitudes. Both Ca2+ spikes were blocked by cobalt (Co2+) but were unaffected by TTX or QX-222. The low-threshold spike (LTS) had a smaller amplitude and inactivated when membrane potential was depolarized past -40 mV or when evoked at a fast rate (greater than 0.5 Hz). The high-threshold spike (HTS) typically had a larger amplitude and longer duration, was not inactivated at potentials which inactivated the LTS, and could be evoked at rates of up to 10 Hz. Single-electrode voltage-clamp analysis revealed that 3 distinct components of the Ca2+ current were present in most cells. From a negative holding potential (-90 mV), 2 separate peak inward currents were observed; a low-threshold transient current, similar to a T-type Ca2+ current, activated at -40 mV, whereas a large-amplitude inactivating current activated above -20 mV. This large inactivating Ca2+ current was significantly inactivated at a holding potential of -40 mV or by brief prepulses to positive potentials, and was similar to an N-type Ca2+ current. A sustained Ca2+ current (L-type) was observed which was not altered by different holding potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polarizing currents increase noradrenaline-elicited accumulation of cyclic AMP in rat cerebral cortex.

Cyclic AMP accumulation elicited by noradrenaline was determined in cerebral cortical slices of rats 24 h after an application of weak anodal direct current (anodal polarization) to the surface of the sensorimotor cortex. Noradrenaline-elicited accumulation of cyclic AMP was altered regionally by the anodal polarization in relation to the duration and intensity of the polarizing current. The cyclic AMP accumulation elicited by noradrenaline was highest in the left anterior cortical region including the polarized point under polarization conditions of 0.3 microA for 1.5 h and 3.0 microA for 30 min. Under these two polarization conditions, the cyclic AMP accumulation elicited by noradrenaline was higher than that in the non-polarized control in the same cortical region. Furthermore, the beta-adrenergic antagonist propranolol almost completely reduced the elicited accumulation of cyclic AMP by noradrenaline to the control level. These results suggest that anodal polarization enhances activity of noradrenaline-sensitive cyclic AMP generating systems through beta-adrenergic mechanisms as a function of both duration and intensity in the cerebral cortex and that one polarization event has a long-lasting aftereffect on noradrenaline-sensitive cyclic AMP generating systems in the cerebral cortex.     

Effects of serotonin on the intrinsic membrane properties of layer II medial entorhinal cortex neurons

Although serotonin (5-HT) is an important neuromodulator in the superficial layers of the medial entorhinal cortex (mEC), there is some disagreement concerning its influences upon the membrane properties of neurons within this region. We performed whole cell recordings of mEC Layer II projection neurons in rat brain slices in order to characterize the intrinsic influences of 5-HT. In current clamp, 5-HT evoked a biphasic response consisting of a moderately short latency and large amplitude hyperpolarization followed by a slowly developing, long lasting, and small amplitude depolarization. Correspondingly, in voltage clamp, 5-HT evoked a robust outward followed by a smaller inward shift of holding current. The outward current evoked by 5-HT showed a consistent current/voltage (I/V) relationship across cells with inward rectification, and demonstrating a reversal potential that was systematically dependent upon the extracellular concentration of K(+), suggesting that it was predominantly carried by potassium ions. However, the inward current showed a less consistent I/V relationship across different cells, suggesting multiple independent ionic mechanisms. The outward current was mediated through activation of 5-HT(1A) receptors via a G-protein dependent mechanism while inward currents were evoked in a 5-HT(1A)-independent fashion. A significant proportion of the inward current was blocked by the I(h) inhibitor ZD7288 and appeared to be due to 5-HT modulation of I(h) as 5-HT shifted the activation curve of I(h) in a depolarizing fashion. Serotonin is thus likely to influence, in a composite fashion, the information processing of Layer II neurons in the mEC and thus, the passage of neocortical information via the perforant pathway to the hippocampus.     

Uncoupling of flow and metabolism induced by sodium nitroprusside in rat cerebral cortex

The effects of sodium nitroprusside (SNP) on cerebral blood flow and glucose metabolism were investigated by the microinfusion of SNP into rat cerebral cortex. A significant enhancement in glucose metabolism, as measured using [14C]deoxyglucose (DG), was observed throughout widespread areas of the cerebral cortex within 1 h of microinjection of 50 nmol/microl SNP. Using a kinetic analysis, the increase in glucose metabolism was found to be due to an increase in the phosphorylation of [14C]DG in the brain. On the other hand, regional cerebral blood flow, as measured using [14C]iodoantypirine, was not significantly altered by the SNP infusion. No significant cell death was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining 1 h after the SNP infusion. The uncoupling of flow and metabolism was almost completely prevented by pretreatment with an NMDA antagonist, MK-801. However, pretreatment with MK-801 did not prevent the SNP-induced neural cell death detected 6 h after the SNP infusion. These results suggest that the SNP-induced uncoupling of flow and metabolism was not directly related to neural cell death in the cerebral cortex.     

The effect of locus ceruleus lesion on water, sodium and potassium content of the rat cerebral cortex

Locus ceruleus lesion decreases the density of ouabain binding sites, and presumably Na+, K+-ATPase, in brain microvessels. To determine if this decrease affects the transport of Na+, K+ or water across the blood-brain barrier, we studied the influence of unilateral locus ceruleus lesion on Na+, K+ and water content of the ipsilateral cerebral cortex. Unilateral locus ceruleus lesion depleted norepinephrine in the ipsilateral cerebral cortex but had no effect on tissue Na+, K+ or water under steady-state conditions. When the Na+/K+ exchange pump of the blood-brain barrier was stressed by hyperkalaemia, K+ content in the ipsilateral cerebral cortex rose to higher levels than in the contralateral cortex, but the difference did not reach statistical significance. Locus ceruleus lesion also did not cause significant differences in the cerebral cortical content of water, Na+ or K+ in hyponatraemia. The results suggest that brain water and ion homeostasis are tightly controlled, probably by multiple mechanisms with biological redundancies, so that even a 50% decrease in the density of ouabain binding sites in brain endothelium does not result in significant alterations in brain water, Na+ or K+ content.     

Cellular properties of principal neurons in the rat entorhinal cortex. II. The medial entorhinal cortex

Principal neurons in different medial entorhinal cortex (MEC) layers show variations in spatial modulation that stabilize between 15 and 30 days postnatally. These in vivo variations are likely due to differences in intrinsic membrane properties and integrative capacities of neurons. The latter depends on inputs and thus potentially on the morphology of principal neurons. In this comprehensive study, we systematically compared the morphological and physiological characteristics of principal neurons in all MEC layers of newborn rats before and after weaning. We recorded simultaneously from up to four post-hoc morphologically identified MEC principal neurons in vitro. Neurons in L(ayer) I-LIII have dendritic and axonal arbors mainly in superficial layers, and LVI neurons mainly in deep layers. The dendritic and axonal trees of part of LV neurons diverge throughout all layers. Physiological properties of principal neurons differ between layers. In LII, most neurons have a prominent sag potential, resonance and membrane oscillations. Neurons in LIII and LVI fire relatively regular, and lack sag potentials and membrane oscillations. LV neurons show the most prominent spike-frequency adaptation and highest input resistance. The data indicate that adult-like principal neuron types can be differentiated early on during postnatal development. The results of the accompanying paper, in which principal neurons in the lateral entorhinal cortex (LEC) were described (Canto and Witter,2011), revealed that significant differences between LEC and MEC exist mainly in LII neurons. We therefore systematically analyzed changes in LII biophysical properties along the mediolateral axis of MEC and LEC. There is a gradient in properties typical for MEC LII neurons. These properties are most pronounced in medially located neurons and become less apparent in more laterally positioned ones. This gradient continues into LEC, such that in LEC medially positioned neurons share some properties with adjacent MEC cells.     

Cellular properties of principal neurons in the rat entorhinal cortex. I. The lateral entorhinal cortex

The lateral entorhinal cortex (LEC) provides a major cortical input to the hippocampal formation, equaling that of the medial entorhinal cortex (MEC). To understand the functional contributions made by LEC, basic knowledge of individual neurons, in the context of the intrinsic network, is needed. The aim of this study is to compare physiological and morphological properties of principal neurons in different LEC layers in postnatal rats. Using in vitro whole cell current-clamp recordings from up to four post hoc morphologically identified neurons simultaneously, we established that principal neurons show layer specific physiological and morphological properties, similar to those reported previously in adults. Principal neurons in L(ayer) I, LII, and LIII have the majority of their dendrites and axonal collaterals alone in superficial layers. LV contains mainly pyramidal neurons with dendrites and axons extending throughout all layers. A minority of LV and all principal neurons in LVI are neurons with dendrites confined to deep layers and axons in superficial and deep layers. Physiologically, input resistances and time constants of LII neurons are lower and shorter, respectively, than those observed in LV neurons. Fifty-four percent of LII neurons have sag potentials, resonance properties, and rebounds at the offset of hyperpolarizing current injection, whereas LIII and LVI neurons do not have any of these. LV neurons show prominent spike-frequency adaptation and a decrease in spike amplitudes in response to strong depolarization. Despite the well-developed interlaminar communication in LEC, the laminar differences in the biophysical and morphological properties of neurons suggest that their in vivo firing patterns and functions differ, similar to what is known for neurons in different MEC layers.     

Ontogeny of thymidine kinase and DNA in the hypothalamus and cerebral cortex of the rat: Effects of neonatal estrogen

Developmental patterns of activity for thymidine kinase and DNA content were determined for the hypothalamus and cerebral cortex of the female rat during the first 3 postnatal weeks. The DNA content and thymidine kinase activity for both brain structures were maximal at ages 1 and 3 days, respectively. Total DNA content for the hypothalamus significantly increased 21% between ages 1 and 21 days. Administration of 17β-estradiol benzoate (EB) during the sexually critical period of brain differentiation had no effect on thymidine kinase in either brain structure. However, a transient and highly significant decrease in DNA content occurred in the hypothalamus of the 2-day-old EB-treated rat. These data indicate that (a) DNA synthesis occurs in the postnatal hypothalamus, (b) DNA synthesis in the hypothalamus and cortex is not tightly coupled to the activity of thymidine kinase and, (c) the EB-induced decrease in hypothalamic DNA is not related to changes in thymidine kinase.     

Development of voltage-dependent calcium, sodium, and potassium currents in Xenopus spinal neurons

Action potentials of embryonic nerve and muscle cells often have a different ionic dependence and longer duration than those of mature cells. The action potential of spinal cord neurons from Xenopus laevis exhibits a prominent calcium component at early stages of development that diminishes with age as the impulse becomes principally sodium dependent. Whole-cell voltage-clamp analysis has been undertaken to characterize the changes in membrane currents during development of these neurons in culture. Four voltage-dependent currents of cells were identified and examined during the first day in vitro, when most of the change in the action potential occurs. There are no changes in the peak density of the calcium current (ICa), its voltage dependence, or time to half-maximal activation; a small increase in inactivation is apparent. The major change in sodium current (INa) is a 2-fold increase in its density. In addition, more subtle changes in the kinetics of the macroscopic sodium current were noted. The peak density of voltage-dependent potassium current (IKv) increases 3-fold, and this current becomes activated almost twice as fast. No changes were noted in the extent of its inactivation. The calcium-dependent potassium current (IKc) consists of an inactivating and a sustained component. The former increases 2-fold in peak current density, and the latter increases similarly at less depolarized voltages. The changes in these currents contribute to the decrease in duration and the change in ionic dependence of the impulse.     

Spontaneous neuronal discharge patterns in developing organotypic mega-co-cultures of neonatal rat cerebral cortex

Sagittal slices of neonatal rat neocortex, extending from the prefrontal to the occipital area, were cultured separately or in pairs, oriented in such a way that axons projecting from the ventricular surface of each explant could innervate the other one. Functional connections were made between as well as within the explants, and spontaneous field potentials and associated action potentials occurred in variable bursts, and with varying degrees of synchrony. Spike-train analysis revealed that the activity patterns seen in these 'mega' co-cultures closely mimic 'tracé alternant' patterns, consisting of trains of burst discharges recurring several times per minute, which are characteristic for the immature intact cerebral cortex during slow-wave sleep. The prefrontal region was consistently less active than the occipital area but the two were qualitatively similar with respect to their patterns of neuronal firing. Isolated mega-cultures, on the other hand, despite their large size, exhibited only intermittent brief bursts that closely resembled those observed both in occipital cortex tissue fragments and in dissociated cell cultures. The mega-co-culture preparation thus appears to give the best currently available approximation to intrinsic cerebral discharge patterns in vivo.