Immature Rat Hippocampus Research Articles

Effect of hypobaric hypoxia on the P2X receptors of pyramidal cells in the immature rat hippocampus CA1 sub‐field

Primary objective: This study was designed to evaluate the effect of hypobaric hypoxia (HH) on the function and expression of P2X receptors in rat hippocampus CA1 pyramidal cells.Research design: The functional changes of P2X receptors were investigated through the cell HH model and the expressional alterations of P2X receptors were observed through the animal HH model.Methods and procedure: P2X receptors mediated currents were recorded from the freshly dissociated CA1 pyramidal cells of 7‐day‐old SD rats by whole cell patch clamp recording. The expression and distribution of P2X receptors were observed through immunohistochemistry and western blot at HH 3‐day and 7‐day.Main outcomes and results: In acute HH conditions, the amplitudes of ATP evoked peak currents were decreased compared to control. The immunohistochemistry and western blot results reflected there was no change in P2X receptors expression after 3 days HH injury, while P2X receptors expression was up‐regulated in response to 7 days HH injury.Conclusions: These findings supported the possibility that the function of P2X receptors was sensitive to HH damage and long‐term function decrease should result in the expression increase of P2X receptors.

Anaesthetic and postanaesthetic effect of isoflurane on the multiple-unit activity of the immature rat hippocampus.

We investigated anesthetic and postanaesthetic effect of isoflurane on the multi-unit activity (MUA) in the CA3 region of immature rat hippocampus. MUA amplitude did not significantly change during application of isoflurane. On the other hand MUA frequency significantly decreased during the anesthesia. After isoflurane discontinuation two phases of MUA frequency recovery were observed: initial rapid increase followed by a slower recovery to the control level. Comparison of recovering period of the receptor mediated systems and spontaneous field activity from isoflurane anesthesia is discussed.

Anaesthetic and Postanaesthetic Effect of Isoflurane on the Multiple-Unit Activity of the Immature Rat Hippocampus

We investigated anesthetic and postanaesthetic effect of isoflurane on the multi-unit activity (MUA) in the CA3 region of immature rat hippocampus. MUA amplitude did not significantly change during application of isoflurane. On the other hand MUA frequency significantly decreased during the anesthesia. After isoflurane discontinuation two phases of MUA frequency recovery were observed: initial rapid increase followed by a slower recovery to the control level. Comparison of recovering period of the receptor mediated systems and spontaneous field activity from isoflurane anesthesia is discussed.

Transcranial direct current stimulation decreases convulsions and spatial memory deficits following pilocarpine-induced status epilepticus in immature rats

Purpose Transcranial direct current stimulation (tDCS) is a recently available, noninvasive brain stimulation technique. The effects of cathodal tDCS on convulsions and spatial memory after status epilepticus (SE) in immature animals were investigated. Methods Rats underwent lithium–pilocarpine-induced SE at postnatal day (P) 20–21 and received daily 30-min cathodal tDCS for 2 weeks at P23-36 through a unilateral epicranial electrode at 200 μA. After tDCS, convulsions over 2 weeks were estimated by 20-h/day video monitoring. The rats were tested in a water maze for spatial learning at P50-53 and the brains were examined for cell loss and mossy fiber sprouting. Results Long-term treatment with weak cathodal tDCS reduced SE-induced hippocampal cell loss, supragranular and CA3 mossy fiber sprouting, and convulsions (reduction of 21%) in immature rats. The tDCS treatment also rescued cognitive impairment following SE. Conclusions These findings suggested that cathodal tDCS has neuroprotective effects on the immature rat hippocampus after pilocarpine-induced SE, including reduced sprouting and subsequent improvements in cognitive performance. Such treatment might also have an antiepileptic effect.

Activity‐ and age‐dependent modulation of GABAergic neurotransmission by System A‐mediated glutamine uptake

GABAergic neurotransmission adapts to maintain normal brain function in a wide range of activity states through multiple mechanisms; pre-synaptic control of quantal size has only recently gained recognition as one of those mechanisms. GABA synthesis from glutamate is coupled with vesicular packaging, and therefore the supply of glutamate can affect inhibitory synaptic strength. Because System A transporters supply glutamine to neurons, where it is converted to glutamate, we hypothesized that regulation of the activity of these transporters could alter glutamine uptake and provide a mechanism to link supply to demand for neurotransmitter GABA. In immature and mature rat hippocampus, after a period of hyperexcitability, we observed a System A-dependent enhancement of inhibitory synaptic strength along with an increase in System A activity in synaptosomes under the same conditions. Under resting conditions, System A's contribution of glutamine to synaptic GABA diminished with age, correlating with reduced SNAT1/SAT1 expression and, even more so, with its activity on synaptic membranes. We conclude that System A activity is highly regulated, by depolarization and developmental cues, to dynamically modulate GABAergic transmission. Our evidence suggests that SNAT1/SAT1 is the transporter that plays a critical role in dynamically modulating inhibition in response to metabolic demands.

Effect of isoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA In the hippocamapus of immature rats

Obiective To investigate the effect ofisoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA in the hippocampus of immature rats.Methods sixty-four 7-clay-old SD rats were randomly assigned into 2 groups(n=32 each):control group(group C)and isoflurane group(group S).group S was exposed to 1.5% isoflurane for 6 h while group C to air.Fore animals were killed before anesthesia(T0,baseline),at 2,4,6 h(T1-3)of isoflurane anesthesia and 4,6,12 and 24 h after anesthesia(T4-7).The hippocampi were immediately removed for determimation of the expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA by RT-PCR.Results Compared with group C,the expression of IL-1β mRNA at T1-5,IL-6 mRNA at T2.3 and TNF-α mRNA at T1-6 in the hippocampus was upregulated in group S.Conclusion The expression of IL-1β mRNA,IL-6 mRNA and TNF-β mRNA was elevated in the hippocampus of immature rats after being exposed to isoflurane. Key words: Isoflurane; Interleukins; Tumour necrosis faetor-alpha

Permissive Role of Insulin in the Expression of Long-Term Potentiation in the Hippocampus of Immature Rats

Many studies indicate that impairment in insulin signaling leads to learning and memory deficits. However, previous studies failed to establish a clear role of insulin in long-term potentiation (LTP), the best cellular model of memory formation. Here we show that while insulin pretreatment did not affect LTP magnitude in the adult rat hippocampus, it facilitated LTP expression in the immature hippocampus. The tyrosine kinase inhibitor AG-1024 abolished the effect of insulin in young rats, suggesting the involvement of the insulin receptor. On the other hand, increasing extracellular glucose concentration failed to facilitate LTP and application of an insulin-responsive glucose transporter-4 inhibitor did not impair the effect of insulin. These results suggest that the facilitatory action of insulin on LTP is not an indirect effect on glucose homeostasis/utilization. Involvement of the MAPK/ERK pathway, a known downstream pathway of insulin signaling, was revealed by pretreatment with PD98059, which blocked the insulin-mediated LTP facilitation. Consistent with this, high-frequency stimulation induced a significant increase in the level of phosphorylated Erk-2 in insulin-treated hippocampus. Taken together, these results suggest that insulin may be an essential factor in the immature brain, allowing the expression of LTP to facilitate learning and memory.

Bumetanide, an NKCC1 Antagonist, Does Not Prevent Formation of Epileptogenic Focus but Blocks Epileptic Focus Seizures in Immature Rat Hippocampus

Excitatory GABA action induced by high [Cl(-)](i) is thought to contribute to seizure generation in neonatal neurons although the mechanism of this effect remains unclear. We report that bumetanide, a NKCC1 antagonist, reduces driving force of GABA-mediated currents (DF(GABA)) in neonatal hippocampal neurons and blocks the giant depolarizing potentials (GDPs), a spontaneous pattern of network activity. In the preparation composed of two intact interconnected hippocampi, bumetanide did not prevent generation of kainate-induced seizures, their propagation to the contralateral hippocampus, and formation of an epileptogenic mirror focus. However, in the isolated mirror focus, bumetanide effectively blocked spontaneous epileptiform activity transforming it to the GDP-like activity pattern. Bumetanide partially reduced DF(GABA) and therefore the excitatory action of GABA in epileptic neurons. Therefore bumetanide is a potent anticonvulsive agent although it cannot prevent formation of the epileptogenic mirror focus. We suggest that an additional mechanism other than NKCC1-mediated contributes to the persistent increase of DF(GABA) in epileptic neurons.

Mechanism of antiseizure effect of isoflurane in the immature rat hippocampus

The volatile anesthetic isoflurane is often used in children in the management of refractory status epilepticus. However the mechanism of anticonvulsant action of isoflurane during early brain development is not clear. In this study we explore the role of excitatory and inhibitory conductances in antiseizure effect of isoflurane using combination of whole-cell patch-clamp and extracellular field potential recording techniques on two models of epilepsy in a hippocampal slice preparation from immature rat. Our data demonstrated that decreasing of excitatory sy-naptic transmission does not account for antiseizure effect of this volatile anesthetic agent. Isoflurane decreases the synchro­nization of neuronal activity mainly through the enhancing of GABAergic inhibition by influencing both phasic and tonic chloride conductances.

Differential Expression of Activating Transcription Factor-2 and c-Jun in the Immature and Adult Rat Hippocampus Following Lithium-Pilocarpine Induced Status Epilepticus

PurposeLithium-pilocarpine induced status epilepticus (LPSE) causes selective and age-dependent neuronal death, although the mechanism of maturation-related injury has not yet been clarified. The activating transcription factor-2 (ATF-2) protein is essential for the normal development of mammalian brain and is activated by c-Jun N-terminal kinase (JNK). It induces the expression of the c-jun gene and modulates the function of the c-Jun protein, a mediator of neuronal death and survival. Therefore, we investigated the expression of c-Jun and ATF-2 protein in the immature and adult rat hippocampus to understand their roles in LPSE-induced neuronal death.Materials and MethodsLithium chloride was administrated to P10 and adult rats followed by pilocarpine. Neuronal injury was assessed by silver and cresyl violet staining, performed 72 hours after status epilepticus. For evaluation of the expression of ATF-2 and c-Jun by immunohistochemical method and Western blot, animals were sacrificed at 0, 4, 24, and 72 hours after the initiation of seizure.ResultsNeuronal injury and expression of c-Jun were maturation-dependently increased by LPSE, whereas ATF-2 immunoreactivity decreased in the mature brain. Since both c-Jun and ATF-2 are activated by JNK, and targets and competitors in the same signal transduction cascade, we could speculate that ATF-2 may compete with c-Jun for JNK phosphorylation.ConclusionThe results suggested a neuroprotective role of ATF-2 in this maturation-related evolution of neuronal cell death from status epilepticus.

Mechanisms of hyperthermia‐induced depression of GABAergic synaptic transmission in the immature rat hippocampus

Clinical observations and experimental studies have shown that hyperthermia can provoke febrile seizures, which are the most common type of pathological brain activity in children. We previously demonstrated that hyperthermia produced a depression of GABAergic neurotransmission in the hippocampus of immature rats in vitro. To investigate the possible mechanisms through which hyperthermia may modulate GABAergic neurotransmission in the hippocampus, whole-cell voltage clamp recordings were performed on CA1 pyramidal neurons in the immature rat brain slices. We found that hyperthermia (38.4-40 degrees C) when compared with baseline temperature of 32 degrees C reduced the frequency of both spontaneous inhibitory post-synaptic currents (sIPSCs) and miniature IPSCs (mIPSCs). Also, hyperthermia decreased the amplitudes of mIPSCs and reduced the mIPSC decay time constants and charge transfer. Non-stationary noise analysis of mIPSCs suggested that the number of open post-synaptic receptors but not single channel conductance was reduced during hyperthermia. Activation of adenylyl cyclase with forskolin prevented, whereas protein kinase A inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide potentiated, the hyperthermia (40 degrees C)-induced depression of evoked IPSCs (evIPSCs). But protein kinase C activator phorbol 12, 13-dibutyrate (PDBu) did not significantly affect this depression of evIPSCs induced by hyperthermia. Furthermore, hyperthermia-induced depression of evIPSCs was attenuated by 4-aminopyridine, but not by BaCl(2). These results suggest that hyperthermia reduces GABA release from pre-synaptic terminals, in part by blocking the adenylyl cyclase-protein kinase A signaling pathway and activating pre-synaptic 4-aminopyridine-sensitive K(+) channels. Also, the changes in amplitude and decay time constant of the mIPSCs may suggest that hyperthermia also decreases post-synaptic GABA(A) receptor function.

Receptor‐specific inhibition of GABAB‐activated K+ currents by muscarinic and metabotropic glutamate receptors in immature rat hippocampus

It has been shown that the activation of G(q)-coupled receptors (G(q)PCRs) in cardiac myocytes inhibits the G protein-gated inwardly rectifying K(+) current (I(GIRK)) via receptor-specific depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)). In this study, we investigated the mechanism of the receptor-mediated regulation of I(GIRK) in acutely isolated hippocampal CA1 neurons by the muscarinic receptor agonist, carbachol (CCh), and the group I metabotropic glutamate receptor (mGluR) agonist, 3,5-dihydroxyphenylglycine (DHPG). I(GIRK) was activated by the GABA(B) receptor agonist, baclofen. When baclofen was repetitively applied at intervals of 2-3 min, the amplitude of the second I(GIRK) was 92.3 +/- 1.7% of the first I(GIRK) in control. Pretreatment of neurons with CCh or DHPG prior to the second application of baclofen caused a reduction in the amplitude of the second I(GIRK) to 54.8 +/- 1.3% and 51.4 +/- 0.6%, respectively. In PLCbeta1 knockout mice, the effect of CCh on I(GIRK) was significantly reduced, whereas the effect of DHPG remained unchanged. The CCh-mediated inhibition of I(GIRK) was almost completely abolished by PKC inhibitors and pipette solutions containing BAPTA. The DHPG-mediated inhibition of I(GIRK) was attenuated by the inhibition of phospholipase A(2) (PLA(2)), or the sequestration of arachidonic acid. We confirmed that DHPG eliminated the inhibition of I(GIRK) by arachidonic acid. These results indicate that muscarinic inhibition of I(GIRK) is mediated by the PLC/PKC signalling pathway, while group I mGluR inhibition of I(GIRK) occurs via the PLA(2)-dependent production of arachidonic acid. These results present a novel receptor-specific mechanism for crosstalk between G(q)PCRs and GABA(B) receptors.

Changes in microtubule-associated protein-2 (MAP2) expression during development and after status epilepticus in the immature rat hippocampus

In this study, we analyzed the spatiotemporal expression patterns of the high-molecular weight (MAP2a and b) and low-molecular weight (MAP2c and d) cytoskeletal microtubule-associated protein-2 (MAP2) isoforms with Western blotting, and the cellular localization of the high-molecular weight MAP2 isoforms with immunocytochemistry in the hippocampi of 1- to 21-day-old rats. Moreover, the temporal profile (from 30min to 1 week) of MAP2 isoform reactivity to kainic acid-induced status epilepticus was studied in P9 rats. During development, the expression of the high-molecular weight MAP2 isoforms significantly increased, while the low-molecular weight isoforms decreased, the most prominent changes occurring during the second postnatal week. This developmental increase in the high-molecular weight MAP2 expression was also confirmed with immunocytochemistry, which showed increased immunoreactivity, particularly in the molecular layers of the dentate gyrus, and in CA1 and CA3 stratum radiatum. In 9-day-old rats, status epilepticus resulted in a rapid transient increase (about 210%) in the high-molecular weight MAP2 expression, without any effect on the low-molecular weight MAP2. Moreover, disturbed dendritic structure in the CA1 and CA3 stratum radiatum was manifested as formation of varicosities 3h after the kainic acid treatment. The strictly developmentally regulated MAP2 isoform expression suggests different functional roles for these proteins during the postnatal development in the rat hippocampus. Moreover, high-molecular weight MAP2s may play a role in nerve cell survival during cell stress.

Hypoosmolar conditions reduce extracellular volume fraction and enhance epileptiform activity in the CA3 region of the immature rat hippocampus

The osmolarity of the extracellular space (ECS) compartment is an important factor determining the excitability of neuronal tissue. In the adult hippocampus an important role of osmolarity and ECS diffusion parameters on the susceptibility to epileptic events is well established, but the influence of hypo- and hyperosmolar conditions on the immature hippocampus remains elusive. To investigate the influence of osmolarity on epileptiform activity, extracellular field potentials were recorded in the CA3 region of hippocampal slices of immature (postnatal days 4-7) Wistar rats. The ECS diffusion parameters were determined by the real-time tetramethylammonium (TMA+) iontophoretic method with ion-selective microelectrodes in immature hippocampal slices and showed a lack of diffusion anisotropy; a tortuosity of about 1.39; and a volume fraction, alpha, of 0.41 +/- 0.01 (n = 10 slices). A reduction in osmolarity of -90 mOsm induced a decrease in alpha to 0.17 +/- 0.02 (n = 4 slices). The frequency of epileptiform activity elicited in 10-50 microM 4-AP-containing low-Mg2+ solution was increased under -90 mOsm and -40 mOsm hypoosmolar conditions by 39.9% +/- 8.1% (n = 16) and 24.1% +/- 4.8% (n = 10), respectively, whereas hyperosmolar solutions decreased the frequency. A -90-mOsm reduction in the osmolarity of low-Mg2+ solution induced epileptiform activity in nine of 19 slices. In summary, these results demonstrate that hypoosmolar conditions increased excitability and susceptibility to epileptiform activity in immature hippocampal slices, suggesting a functional role of the larger alpha in suppression of seizures.

Lead stimulates ERK1/2 and p38 MAPK phosphorylation in the hippocampus of immature rats

Lead (Pb 2+) is widely recognized as a neurotoxicant whose mechanisms of action are not completely established. We have previously demonstrated that Pb 2+ can activate the p38 MAPK pathway and increase the phosphorylation of Hsp27 in bovine adrenal chromaffin cells and human SH SY5Y cells over a short incubation period (1 h). In the present work we analyzed the effects of Pb 2+ administered in vivo on the level and the phosphorylation state of ERK1/2 and p38 MAPK in the hippocampus of immature rats. Rats were treated with lead acetate (2, 8 or 12 mg/kg, i.p.) or saline (control) over the 8th to 12th postnatal days, and hippocampal slices were prepared on the14th day. The Pb 2+ level in the lead-treated animals increased 2.5–6-fold in the blood (3.0–6.0 μg/dl) and 2.0–3.0-fold in the forebrain (78–103 ng/g wet weight), compared to control (saline). The phosphorylation of both ERK1/2 and p38 MAPK was significantly increased by prior exposure to Pb 2+ in vivo. In in vitro experiments, hippocampal slices from 14-day-old rats were exposed to Pb 2+ (1–10 μM) for 1 and 3 h. There were no changes in the phosphorylation state of ERK and p38 MAPK for 1-h incubation, whereas a significant increase of ERK1/2 and p38 MAPK phosphorylation by Pb 2+ (5 μM) was observed for the 3-h incubation. Cell viability measured using MTT was not modified in any of the conditions tested. These results indicate that the phosphorylation of hippocampal ERK1/2 and p38 MAPK is stimulated by lead in a period of rapid brain development, an effect that may underlie, at least in part, the neurotoxicty elicited by this metal.

Neuronal cell death in hippocampus induced by homocysteic acid in immature rats.

To examine the morphologic alterations in the cerebral cortex and hippocampus of immature rats 6 days after the generalized clonic-tonic seizures induced by homocysteic acid (HCA). Seizures were induced by bilateral intracerebroventricular infusion of HCA (600 nmol per each side) in 12-day-old rats. After 6 days, rat pups were transcardially perfused under deep ether anesthesia with heparinized normal saline and subsequently with the fixation solution (4% paraformaldehyde in phosphate buffer, pH 7.4, for light microscopy) or with Karnovsky's solution (4% paraformaldehyde and 2% glutaraldehyde in phosphate buffer, pH 7.4, for electron microscopic analysis). Nissl stain and the DNA-specific dye bis-benzimide (Hoechst 33342) were used. No pathologic changes were found in the cerebral cortex, whereas serious alterations occurred in the hippocampus. A total loss of CA3 pyramidal cells was observed, with marked changes in the CA1 region and dentate gyrus. A prominent glial reaction was seen in many regions of the hippocampal formation. A slight dilatation of the cerebral ventricles was noticed in some experimental as well as control animals. In the granule cell layer of the dentate gyrus, neurons with segmented or fragmented nuclei in various stages of degeneration were detected, displaying the features of apoptotic death. These findings demonstrate the vulnerability of the immature rat brain, which most likely reflects both the direct neurotoxic effect of HCA and prolonged seizure activity. The relative contribution of these two factors still remains to be assessed.

Developmental patterns and plasticities: the hippocampal model

Because developmental activity-dependent synaptic plasticity has been hypothesized to participate in network refinement, leading to the precise mapping of synaptic contacts constituting a functional brain, it is important to investigate the spatio-temporal structure of immature network activities. This article is briefly reviewing 15 years of studies on the immature rat hippocampus which, together with recent results obtained from awake rat pups, represent an important step toward the understanding of spontaneous patterns of activity and their potential implication in network maturation. Due to synergistic excitatory actions of GABA and glutamate receptor mediated signals during early postnatal life, spontaneous patterns of hippocampal activity that have been characterized both in vitro and in vivo are likely to provide hebbian modulation of developing glutamatergic and GABAergic synapses. Together with studies on trophic actions of these transmitters, study of the immature hippocampal network patterns and plasticities allows for multiple technical and conceptual approaches and represents an interesting experimental model for development studies.

Decreased Glutamate Receptor 2 Expression and Enhanced Epileptogenesis in Immature Rat Hippocampus after Perinatal Hypoxia-Induced Seizures

Hypoxic encephalopathy is the most common cause of neonatal seizures and can lead to chronic epilepsy. In rats at postnatal days 10-12 (P10-12), global hypoxia induces spontaneous seizures and chronically decreases seizure threshold, thus mimicking clinical aspects of neonatal hypoxia. We have shown previously that the acute and chronic epileptogenic effects of hypoxia are age-dependent and require AMPA receptor activation. In this study, we aimed to determine whether hypoxia-induced seizures and epileptogenesis are associated with maturational and seizure-induced changes in AMPA receptor composition and function. Northern and Western blots indicated that glutamate receptor 2 (GluR2) mRNA and protein expression were significantly lower in neocortex and hippocampus at P10-12 compared with adult. After hypoxia-induced seizures at P10, GluR2 mRNA was significantly decreased within 48 hr, and GluR2 protein was significantly decreased within 96 hr. AMPA-induced Co(2+) uptake by neurons in hippocampal slices indicated higher expression of Ca(2+)-permeable AMPA receptors in immature pyramidal neurons compared with adult. In slices obtained 96 hr after hypoxia-induced seizures, AMPA-induced Co(2+) uptake was significantly increased compared with age-matched controls, and field recordings revealed increased tetanus-induced afterdischarges that could be kindled in the absence of NMDA receptor activation. In situ end labeling showed no acute or delayed cell death after hypoxia-induced seizures. Our results indicate that susceptibility to hypoxia-induced seizures occurs during a developmental stage in which the expression of Ca(2+)-permeable AMPA receptors is relatively high. Furthermore, perinatal hypoxia-induced seizures induce increased expression of Ca(2+)-permeable AMPA receptors and an increased capacity for AMPA receptor-mediated epileptogenesis without inducing cell death.

Persistently modified h-channels after complex febrile seizures convert the seizure-induced enhancement of inhibition to hyperexcitability

Febrile seizures are the most common type of developmental seizures, affecting up to 5% of children. Experimental complex febrile seizures involving the immature rat hippocampus led to a persistent lowering of seizure threshold despite an upregulation of inhibition. Here we provide a mechanistic resolution to this paradox by showing that, in the hippocampus of rats that had febrile seizures, the long-lasting enhancement of the widely expressed intrinsic membrane conductance Ih converts the potentiated synaptic inhibition to hyperexcitability in a frequency-dependent manner. The altered gain of this molecular inhibition-excitation converter reveals a new mechanism for controlling the balance of excitation-inhibition in the limbic system. In addition, here we show for the first time that h-channels are modified in a human neurological disease paradigm.

Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation.

Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network.