Lacunosum Moleculare Research Articles

Prospects of Teaching in Pathology 2, Abstract 151153, Symposium

The functions of vascular endothelial growth factor C (VEGF-C) and the VEGF receptor 3 (VEGFR-3) in the nervous system are not well known. In this study, we examined the role of VEGF-C and VEGFR-3 in ischemic preconditioning (IPC)-induced tolerance in the mouse hippocampus. Adult male C57BL/6 mice were subjected to either severe ischemia (SI) induced by 40 min of bilateral common carotid artery occlusion (BCCAO) with or without IPC (5-min BCCAO) or IPC only. Cerebral blood flow was measured during ischemic periods using laser Doppler flowmetry. Neuronal damage was assessed histologically, and VEGF-C and VEGFR-3 expression levels were assessed through immunostaining. Fluoro-Jade B-labeled cells were abundant in the CA1 area 7 days after SI without IPC (sham + SI group), whereas cells were rarely labeled in mice subjected to IPC followed by SI (IPC + SI group). Similarly, the number of neuronal nuclei (NeuN)-positive cells in the CA1 area was significantly lower in the sham + SI group than in the IPC + SI group. Interestingly, we found that sublethal IPC treatment induced prominent VEGF-C expression in the CA1 pyramidal neurons and VEGFR-3 expression in the stratum radiatum and stratum lacunosum moleculare after 3 days of reperfusion that were sustained for 7 days. Moreover, VEGF-C immunoreactivity was also markedly increased, whereas VEGFR-3 expression was sustained in tolerance-acquired CA1 neurons after SI. Application of a VEGFR-3 inhibitor, SAR131675, abolished the IPC-induced neuroprotection in a dose-dependent manner in the mouse hippocampus. These results suggest that VEGF-C/VEGFR-3 signaling is associated with IPC-induced hippocampal tolerance to lethal ischemia.

Mapping cerebral glucose metabolism during spatial learning: interactions of development and traumatic brain injury.

Previous studies have demonstrated that, compared to adults, postnatal day 17 (P17) and P28 rats show remarkable cognitive recovery in the Morris water maze (MWM) following fluid percussion injury (FPI). This observed age-at-trauma effect could result from either younger animals solving the MWM task using noninjured neural circuitry or an inability of adult and P28 brains to activate appropriate neural networks due to trauma-induced neurological dysfunction. To address these possibilities, we compared "activated" brain regions during normal MWM acquisition and following FP injury. To generate "activated" images of the brain while animals were performing the MWM task, qualitative [14C]2-deoxy-D-glucose was conducted on days 2, 5, and 14 during training in sham and injured adult, P28, and P17 rats. When maturational changes in cerebral glucose metabolism are taken into account, the results suggests similar activity changes in the cerebral cortex and lacunosum moleculare of CA1 during acquisition in all age groups, suggesting that the developmental rates of MWM learning do not correspond to different patterns of activated cerebral metabolism. Injured P17s, showing no latency deficits, revealed activated cerebral metabolic patterns similar to noninjured P17 animals. In P28 and adult cases, animals exhibited cognitive deficits and their metabolic studies indicated that the cortical-hippocampal pattern of activation was disrupted by marked injury-induced metabolic depression, which primarily affected the ipsilateral hemisphere and lasted for as long as 14 days in adult animals.

Nicotine at concentrations found in cigarette smokers activates and desensitizes nicotinic acetylcholine receptors in CA1 interneurons of rat hippocampus

Behavioral effects of cigarette smoking are attributed to the interactions of nicotine with brain nicotinic acetylcholine receptors (nAChRs). However, the mechanisms by which nAChR function in developing and mature brain is affected by a smoker's level of nicotine (50–500 nM) remain unclear. Thus, the objective of this study was to determine the concentration- and time-dependent effects of nicotine on α7 and α4β2 nAChRs, the two major brain subtypes, natively expressed in CA1 interneurons of rat hippocampal slices. Only at concentrations ≥5 μM did nicotine (applied for 6–60 s) elicit action potentials or measurable whole-cell currents (EC 50=158 μM) in stratum radiatum interneurons that express α7 nAChRs. Continuous exposure for 10–15 min of the neurons to nicotine (0.5–2.5 μM) inhibited α7 nAChR-mediated currents (IC 50=640 nM) evoked by choline (10 mM). Nicotine (≥0.125 μM) applied to the neurons for 1–5 min induced slowly desensitizing whole-cell currents (EC 50=3.2 μM) in stratum lacunosum moleculare interneurons; this effect was mediated by α4β2 nAChRs. Also via activation of α4β2 nAChRs, nicotine (0.125–0.5 μM) increased the frequency and amplitude of GABAergic postsynaptic currents (PSCs) in stratum radiatum interneurons. However, exposure of the neurons for 10–15 min to nicotine (0.25–0.5 μM) resulted in desensitization of α4β2 nAChRs. It is suggested that nanomolar concentrations of nicotine after acute intake suppress inhibitory inputs to pyramidal cells through a disinhibitory mechanism involving activation of α4β2 nAChRs and desensitization of α7 nAChRs, and after chronic intake leads to up-regulation of both receptor subtypes via desensitization. These findings have direct implications to the actions of nicotine in cigarette smokers.

Activity-dependent presynaptic autoinhibition by group II metabotropic glutamate receptors at the perforant path inputs to the dentate gyrus and CA1

Pharmacological activation of metabotropic glutamate receptors (mGluRs) can inhibit synaptic transmission; however, relatively little evidence exists regarding the physiological conditions under which such autoreceptors are activated by synaptically released glutamate. Bath application of selective group II mGluR agonists profoundly inhibited field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the perforant path inputs to both the mid-molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1. Application of the group II selective mGluR antagonist LY341495 resulted in an increase in the relative amplitude of a test fEPSP evoked 200 ms after a conditioning burst, but not after a single conditioning stimulus, in both pathways. Antagonist application also resulted in a marked increase in the relative amplitude of test population spikes evoked in the dentate gyrus following a conditioning burst. These observations are consistent with a presynaptic autoinhibitory action of group II metabotropic receptors that is revealed following burst stimulation of the pathway, consistent with their localisation in the preterminal zone. Activation of group II mGluRs during theta–gamma pattern discharge of projection neurones in the entorhinal cortex is likely to play an important role in the regulation of synaptic transmission and plasticity in the perforant pathway.

Circuit-Specific Alterations in Hippocampal Synaptophysin Immunoreactivity Predict Spatial Learning Impairment in Aged Rats

The present study examined the long-standing concept that changes in hippocampal circuitry contribute to age-related learning impairment. Individual differences in spatial learning were documented in young and aged Long-Evans rats by using a hippocampal-dependent version of the Morris water maze. Postmortem analysis used a confocal laser-scanning microscopy method to quantify changes in immunofluorescence staining for the presynaptic vesicle glycoprotein, synaptophysin (SYN), in the principal relays of hippocampal circuitry. Comparisons based on chronological age alone failed to reveal a reliable difference in the intensity of SYN staining in any region that was examined. In contrast, aged subjects with spatial learning deficits displayed significant reductions in SYN immunoreactivity in CA3 lacunosum-moleculare (LM) relative to either young controls or age-matched rats with preserved learning. SYN intensity values for the latter groups were indistinguishable. In addition, individual differences in spatial learning capacity among the aged rats correlated with levels of SYN staining selectively in three regions: outer and middle portions of the dentate gyrus molecular layer and CA3-LM. The cross-sectional area of SYN labeling, by comparison, was not reliably affected in relation cognitive status. These findings are the first to demonstrate that a circuit-specific pattern of variability in the connectional organization of the hippocampus is coupled to individual differences in the cognitive outcome of normal aging. The regional specificity of these effects suggests that a decline in the fidelity of input to the hippocampus from the entorhinal cortex may play a critical role.

Input-Specific Immunolocalization of Differentially Phosphorylated Kv4.2 in the Mouse Brain

Voltage-gated A-type potassium channels such as Kv4.2 regulate generation of action potentials and are localized abundantly in the hippocampus and striatum. Phosphorylation consensus sites for various kinases exist within the sequence of the potassium channel subunit Kv4.2, including consensus sites for extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK), protein kinase A (PKA), protein kinase C (PKC), and calcium/calmodulin-dependent kinase II (CaMKII), and kinase assays have shown that particular amino acids of the consensus sites are bonafide phosphorylation sites in vitro. We have developed antibodies recognizing Kv4.2 triply phosphorylated at the three ERK sites as well as two antibodies recognizing singly phosphorylated Kv4.2 channels at the PKA sites (one amino-terminal and one carboxy-terminal). In the present study, we report the development of reliable immunohistochemistry protocols to study the localization of these phosphorylated versions of Kv4.2, as well as total Kv4.2 in the mouse brain. A general description of the areas highlighted by these antibodies includes the hippocampus, amygdala, cortex, and cerebellum. Such areas display robust synaptic plasticity and have been implicated in spatial, associative, and motor learning. Interestingly, in the hippocampus, the antibodies to differentially phosphorylated Kv4.2 channels localize to specific afferent pathways, indicating that the Kv4.2 phosphorylation state may be input specific. For example, the stratum lacunosum moleculare, which receives inputs from the entorhinal cortex via the perforant pathway, displays relatively little ERK-phosphorylated Kv4.2 or PKA carboxy-terminal-phosphorylated Kv4.2. However, this same layer is highlighted by antibodies that recognize Kv4.2 that has been phosphorylated by PKA at the amino terminus. Similarly, of the three antibodies tested, the soma of CA3 neurons are primarily recognized by the ERK triply phosphorylated Kv4.2 antibody, and the mossy fiber inputs to CA3 are primarily recognized by the carboxy-terminal PKA-phosphorylated Kv4.2. This differential phosphorylation is particularly interesting in two contexts. First, phosphorylation may be serving as a mechanism for targeting. For example, the amino-terminal PKA phosphorylation may be acting as a tag for a discrete pool of Kv4.2 to enter stratum lacunosum moleculare. Second, as phosphorylation may regulate channel biophysical properties, differential phosphorylation of Kv4.2 in the dendrites of pyramidal neurons may confer unique biophysical properties upon particular dendritic input layers.

Hereditary Neuronal Microdysgenesis and Acquired Lesions in the Hippocampal Formation of Ihara Epileptic Rat

Purpose: Ccrebral dysplasia including neuronal microdysgcnesis is reported in a large series or cortical resections obtained from patients with medically intractable epilepsy, Some investigators believe that the cerebral dysplasia is linked intiinatcly to epileptogcnesis and seizurepropagation. The Ihara Epileptic Rat (IER), previously tcrmed Ihara's genetically epilcptic rat (IGER), is a novel rat mutant with limbic‐like seizures. Hyperactivity with wild running, jumping, kicking, or wet‐ dog shaking appear at approximately 2 months after birth. Abnormal circling seizures begin approximately 3 months after hirth, and the rats repeatcdly rotate, as if chasing their tails. Spontaneous generalized tonic‐chic convulsions become evident approximately 5–6 months after birth. The frequency of the generalized convulsions usually is no more than I attack per week initially, but with aging thc convulsions become more frequent. Seizure forms present i n I of 3 patterns: abnormal behavior, circling seizures, and generalized tonic‐clonic coilvulsions, which can be documented by automatic seimre monitoring systems. Neuropathological findings in the hippocampus or IER include cctopic cell clusters consisting of neurons in the region of the Ftratum radiatum, disorientation oi pyramidal neuron alignment. a “wavy” granule cell layer, and enlargement of the hippocampal formation. It is assumed that the changes in the hippocampus consist of hereditary lesions and acquired lesions. The aim of the present study was to distinguish the hereditary hippocampal lesions from the acquired lesion in the IER model. Methods: Two month‐old IERs without apparent abnormal behavior, and twelve month‐old IERs with abnormal behaviors, abnormal circling seizures, or generalized tonic‐clonic convulsions (5 males and 5 fernales pel‐ group) were used for neuropiithological investigation. Agematchcd Wistar rats were used as controls. Anesthetized rats were pcrlused with 4% formaldehyde injected into the heart, and the brains then wcrc removed. Frozen brains were serially sliced 30 pm with a frozcii inicrotome and thc slices stained with Cresyl violet acetate, Nissl staining. Histopathological studies were performcd i n order to examine the hippocampal lesions, the features, the localization, and the sevcrity. Semiquantitativc analysis lor these lesions was also carried out. Results: Ectopic neuronal clusters i n the region of the stratum EIdiatum on the border of stratum lacunosum moleculare in the CAI of the anterior dorsal hippocampus occurred without exception in male and fcmale 2 and 12 month‐old IER. Disarray of pyramidal neurons or sinall foci of ncuronal interruption in the zonal structure of the pyra‐ midal ccll layer was also found, almost always in the CAI and sometimes in the CA3 regions. These lesions, neuronal clusters, and disorientation of pyramidal neurons were coininoii findings in all IER examined. Conversely, the wavy forin of the dentate gyrus and enlargement of the hippocainpal formation, which occurred in thc I2 month‐old rats, showed differences iii the severity among the groups of IER. Thc degree of wavy form or enlargement of hippocampal forniation dcpcndcd on the degree of thc seizure activities. The degree of lesions was more scvcre in the hippocampus in rats with more severe seizure activitics. A mild degree of wavy forin in the granule cell layer, but no enlargement of hippocampal formation, was found in 2‐month old IER. No ectopic neuronal clusters, disarrangement of pyramidal neurons in the hippocainpal formation, wavy form of the granule cell layer, or enlargcmcnt of hippocampus wcre found in any control rats. Conclusions: This study dcmonstrates that the ectopic neuronal clusind disarray of the pyramidal neurons arc hereditary lesions, such as programmed neuronal microdysgenesis. Thc wavy form of the dcntare granule ccll and enlargement of thc hippocampal formation, on the consequence of epileptic activities.

Neuropeptides in hippocampus and cortex in transgenic mice overexpressing V717F β-amyloid precursor protein — initial observations

Immunohistochemistry was used to analyse 18- and 26-month-old transgenic mice overexpressing the human β-amyloid precursor protein under the platelet-derived growth factor-β promoter with regard to presence and distribution of neuropeptides. In addition, antisera/antibodies to tyrosine hydroxylase, acetylcholinesterase, amyloid peptide, glial fibrillary acidic protein and microglial marker OX42 were used. These mice have been reported to exhibit extensive amyloid plaques in the hippocampus and cortex [Masliah et al. (1996) J. Neurosci.16, 5795–5811].The most pronounced changes were related to neuropeptides, whereas differences between wild-type and transgenic mice were less prominent with regard to tyrosine hydroxylase and acetylcholinesterase. The main findings were of two types; (i) involvement of peptide-containing neurites in amyloid β-peptide positive plaques, and (ii) more generalized changes in peptide levels in specific layers, neuron populations and/or subregions in the hippocampal formation and ventral cortices. In contrast, the parietal and auditory cortices were comparatively less affected. The peptide immunoreactivities most strongly involved, both in plaques and in the generalized changes, were galanin, neuropeptide Y, cholecystokinin and enkephalin.This study shows that there is considerable variation both with regard to plaque load and peptide expression even among homozygotes of the same age. The most pronounced changes, predominantly increased peptide levels, were observed in two 26-month-old homozygous mice, for example, galanin-, enkephalin- and cholecystokinin-like immunoreactivities in stratum lacunosum moleculare, and galanin, neuropeptide Y, enkephalin and dynorphin in mossy fibers. Many peptides also showed elevated levels in the ventral cortices. However, decreases were also observed. Thus, galanin-like immunoreactivity could not any longer be detected in the diffusely distributed (presumably noradrenergic) fiber network in all hippocampal and cortical layers, and dynorphin-like immunoreactivity was decreased in stratum moleculare, cholecystokinin-like immunoreactivity in mossy fibers and substance P-like immunoreactivity in fibers around granule cells.The significance of generalized peptide changes is at present unclear. For example, the increase in the mainly inhibitory peptides galanin, neuropeptide Y, enkephalin and dynorphin and the decrease in the mainly excitatory peptide cholecystokinin in mossy fibers (and of substance P fibers around granule cells) indicate a shift in balance towards inhibition of the input to the CA3 pyramidal cell layer. Moreover, it may be speculated that the increase in levels of some of the peptides represents a reaction to nerve injury with the aim to counteract, in different ways, the consequences of injury, for example by exerting trophic actions. Further studies will be needed to establish to what extent these changes are typical for Alzheimer mouse models in general or are associated with the V717F mutation and/or the platelet-derived growth factor-β promoter.

Reduced expression of insulin-like growth factor 1 messenger RNA in the hippocampus of aged rats

Adult neurones remain dependent on neurotrophic factors such as insulin-like growth factor 1 (IGF-1) to sustain neuronal viability by maintaining cell phenotype, supporting synaptic plasticity and providing neuroprotective and neuroregenerative mechanisms. A decline in the cellular expression of neurotrophic factors has been speculated to contribute to the age-related changes that occur in the brain, where the hippocampus appears particularly susceptible. Using in situ hybridisation, we have made a detailed comparison of the expression of IGF-1 mRNA in the hippocampal formation between young (6 months) and aged (23 months) rats. IGF-1 mRNA expression was measured from cell populations containing only high density radioactive labelling (>20 grains/cell) to avoid ambiguity of signal. The amount of IGF-1 mRNA signal was significantly lower in cells of the alveus ( P<0.05) and stratum lacunosum moleculare ( P<0.01) of aged compared to young rats. These findings challenge reports that IGF-1 mRNA is unaltered in the ageing hippocampus and provide further evidence that changes in the IGF-1 system is a significant factor in the progressive age-related deterioration of normal neuronal function.

Properties of a calcium-activated K(+) current on interneurons in the developing rat hippocampus.

Calcium-activated potassium currents have an essential role in regulating excitability in a variety of neurons. Although it is well established that mature CA1 pyramidal neurons possess a Ca(2+)-activated K(+) conductance (I(K(Ca))) with early and late components, modulation by various endogenous neurotransmitters, and sensitivity to K(+) channel toxins, the properties of I(K(Ca)) on hippocampal interneurons (or immature CA1 pyramidal neurons) are relatively unknown. To address this problem, whole-cell voltage-clamp recordings were made from visually identified interneurons in stratum lacunosum-moleculare (L-M) and CA1 pyramidal cells in hippocampal slices from immature rats (P3-P25). A biphasic calcium-activated K(+) tail current was elicited following a brief depolarization from the holding potential (-50 mV). Analysis of the kinetic properties of I(K(Ca)) suggests that an early current component differs between these two cell types. An early I(K(Ca)) with a large peak current amplitude (200.8 +/- 13.2 pA, mean +/- SE), slow time constant of decay (70.9 +/- 3.3 ms), and relatively rapid time to peak (within 15 ms) was observed on L-M interneurons (n = 88), whereas an early I(K(Ca)) with a small peak current amplitude (112.5 +/- 7.3 pA), a fast time constant of decay (39.4 +/- 1.6 ms), and a slower time-to-peak (within 26 ms) was observed on CA1 pyramidal neurons (n = 85). Removal of extracellular calcium or addition of inorganic Ca(2+) channel blockers (cadmium, nickel, or cobalt) was used to demonstrate the calcium dependence of these currents. Addition of norepinephrine, carbachol, and a variety of channel toxins (apamin, iberiotoxin, verruculogen, paxilline, penitrem A, and charybdotoxin) were used to further distinguish between I(K(Ca)) on these two hippocampal cell types. Verruculogen (100 nM), carbachol (100 microM), apamin (100 nM), TEA (1 mM), and iberiotoxin (50 nM) significantly reduced early I(K(Ca)) on CA1 pyramidal neurons; early I(K(Ca)) on L-M interneurons was inhibited by apamin and TEA. Combined with previous work showing that the firing properties of hippocampal interneurons and pyramidal cells differ, our kinetic and pharmacological data provide strong support for the hypothesis that different types of Ca(2+)-activated K(+) current are present on these two cell types.

Central 5-HT 1 and 5-HT 2 binding sites in transgenic mice with reduced glucocorticoid receptor number

Transgenic mice bearing a transgene coding for a glucocorticoid receptor antisense mRNA, which partially blocks glucocorticoid receptor expression, were used in order to clarify the role of glucocorticoid receptors in the regulation of 5-HT 1A, 5-HT 1nonA and 5-HT 2 binding sites labelled by quantitative autoradiography in the frontal and prefrontal cortex, striatum, hypothalamus, amygdala and raphe nuclei. We found that 1 nM [ 3H]8-hydroxy-2-[di- N-propylamino]tetralin ([ 3H]8-OH-DPAT) binding to 5-HT 1A sites was decreased in strata oriens (−15.1±3.5%) and radiatum–lacunosum–moleculare (−13.3±4.3%) of the hippocampal CA 3 area, and 2 nM [ 3H]5-hydroxytryptamine binding to 5-HT 1nonA sites in the presence of 100 nM 8-OH-DPAT and mesulergine was decreased in the dorsal subiculum (−17.8±6.9%). By contrast, 5-HT 2 sites labelled by 0.5 nM of (±)-1-(2,5-dimethoxy-4-[ 125I]iodophenyl)-2-aminopropane was increased in the dorsal subiculum (+35.2±11.5%) and CA 2 area (+29.2±11.3%). The observed differences in binding to 5-HT 1 and 5-HT 2 sites were all located in areas of the hippocampus that contain both gluco- and mineralo-corticoid receptors, and no difference was observed in anatomical structures which contain only glucocorticoid receptors. Therefore, it seems that the important factor for the regulation of these 5-HT receptors is the interaction between gluco- and mineralo-corticoid receptors rather than the absolute density of glucocorticoid receptors. These results suggest that some of the alterations of the serotonergic neurotransmission observed in depressed patients might be secondary to an altered glucocorticoid receptor function.

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus following transient global ischemia

To investigate a potential role of ciliary neurotrophic factor (CNTF) in transient global ischemia, we have studied the postischemic regulatory changes in the expression of CNTF and its receptor, the ligand-binding α-subunit (CNTFRα). Immunoblot analysis demonstrated CNTF levels were slightly upregulated already during the first day after ischemia and then increased markedly by more than 10-fold until 2 weeks postischemia. Immunoreactivity for CNTF became detectable 1 day after ischemia and was localized in reactive astrocytes. The intensity of the immunolabeling was maximal in CA1 during the phase of neuronal cell death (days 3–7 postischemia) and in the deafferented inner molecular layer of the dentate gyrus. Upregulation of CNTF expression was less pronounced in CA3 and absent in the stratum lacunosum moleculare and the outer molecular layer of the dentate gyrus and thus did not simply correlate with astroliosis as represented by upregulation of glial fibrillary acidic protein (GFAP). As shown by in situ hybridization, expression of CNTFRα mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Following ischemia, reactive astrocytes, identified by double labeling with antibodies to GFAP, transiently expressed CNTFRα mRNA with a maximum around postischemic day 3. This astrocytic response was most pronounced in CA1 and in the hilar part of CA3. These results show that CNTF and its receptor are differentially regulated in activated astrocytes of the postischemic hippocampus, indicating that they are involved in the regulation of astrocytic responses and the neuronal reorganizations occurring after an ischemic insult.

Parental deprivation induces N-methyl-D-aspartate-receptor upregulation in limbic brain areas of Octodon degus: protective role of the maternal call.

An optimal coordination between parents and their offspring involves a sequence of reciprocal behaviors to ensure the adequate care of the young, which is critical for a healthy physical, emotional, and social development. Parental deprivation, particularly an impaired child-mother attachment, induces lasting changes in emotional as well as in cognitive capacities in later life. We assessed in the South American precocial species, Octodon degus, whether alterations of glutamatergic function of the limbic system induced by parental deprivation may be a neural correlate for such behavioral changes. Further, we analyzed whether the mother's voice can protect from separation-induced changes of brain function. Changes of N-methyl-D-aspartate (NMDA) receptor expression were examined in the following three groups of 2-week-old Octodon degus pups: (I) control animals who remained undisturbed with the family; (II) animals who were repeatedly separated from the family and individually placed in an unfamiliar environment for behavioral analysis (open field); and (III) animals who were treated like the group described under (lI) but were presented with maternal vocalizations during separation. Relative to those in the control group I, the animals in group II showed an upregulation of NMDA receptor density in the (a)anterior cingulate, prelimbic, infralimbic, and anterior insular cortices; (b)CA1/stratum radiatum; (c)CA1/stratum lacunosum moleculare and CA3/stratum radiatum; and (d)in the basomedial amygdaloid nucleus. Presentation of the maternal call during the separation period (group III) suppressed the separation-induced NMDA receptor upregulation in all regions. The results demonstrate that early life events can influence the expression of transmitter receptors and that maternal behavior, acting to control the pup's socio-emotional environment, is a key factor for regulating such developmental events.

Bilateral growth-related protein expression suggests a transient increase in regenerative potential following brain trauma

The potential of mature central nervous system (CNS) neurons to regenerate after injury represents a fundamental issue in neurobiology. The regional expression of proteins associated with axonal elongation, such as microtubule-associated protein 1B (MAP1B), its phosphorylated isoform (MAP1B-P), growth-associated protein 43 (GAP-43), and polysialylated neural cell-adhesion molecule (PSA-NCAM), was examined using immunohistochemistry from 24 hours to 2 months following lateral fluid percussion brain injury of moderate severity (2.4-2.6 atmospheres) in anesthetized rats. Uninjured (control) rats were subjected to anesthesia and surgery without injury or were subjected to anesthesia alone. Within the site of maximal injury, only increases in MAP1B and MAP1B-P were observed. Increased immunoreactivity was observed bilaterally for all growth-related proteins that were evaluated. By 24 hours postinjury, MAP1B and MAP1B-P increased within the cortex (P < 0.01) and the hippocampus (P < 0.001), whereas MAP1B-P also was elevated in the thalamus (P < 0.05). Within the dentate gyrus, increased immunoreactivity was observed for all proteins examined. By 48 hours postinjury, GAP-43 was elevated bilaterally within the inner molecular layers of the dentate gyrus (P < 0.005) and within the stratum lacunosum moleculare (P < 0.01), the stratum radiatum (P < 0. 005), and the stratum oriens (P < 0.05) of the hippocampus. Increased numbers of PSA-NCAM-labeled neurons were observed in the granule cell layers of the dentate gyrus from 48 hours through 2 weeks postinjury (P < 0.0005). The bilateral nature of increased expression of growth-related proteins differs from unilateral patterns of neuronal degeneration previously characterized for the lateral fluid-percussion model of brain injury. Taken together, these results suggest the existence of a temporary posttraumatic state in which the CNS may have increased regenerative potential. Enhancement of such a response may be one therapeutic strategy in treating CNS injury.

The Establishment of GABAergic and Glutamatergic Synapses on CA1 Pyramidal Neurons is Sequential and Correlates with the Development of the Apical Dendrite

We have performed a morphofunctional analysis of CA1 pyramidal neurons at birth to examine the sequence of formation of GABAergic and glutamatergic postsynaptic currents (PSCs) and to determine their relation to the dendritic arborization of pyramidal neurons. We report that at birth pyramidal neurons are heterogeneous. Three stages of development can be identified: (1) the majority of the neurons (80%) have small somata, an anlage of apical dendrite, and neither spontaneous nor evoked PSCs; (2) 10% of the neurons have a small apical dendrite restricted to the stratum radiatum and PSCs mediated only by GABA(A) receptors; and (3) 10% of the neurons have an apical dendrite that reaches the stratum lacunosum moleculare and PSCs mediated both by GABA(A) and glutamate receptors. These three groups of pyramidal neurons can be differentiated by their capacitance (C(m) = 17.9 +/- 0.8; 30.2 +/- 1.6; 43.2 +/- 3.0 pF, respectively). At birth, the synaptic markers synapsin-1 and synaptophysin labeling are present in dendritic layers but not in the stratum pyramidale, suggesting that GABAergic peridendritic synapses are established before perisomatic ones. The present observations demonstrate that GABAergic and glutamatergic synapses are established sequentially with GABAergic synapses being established first most likely on the apical dendrites of the principal neurons. We propose that different sets of conditions are required for the establishment of functional GABA and glutamate synapses, the latter necessitating more developed neurons that have apical dendrites that reach the lacunosum moleculare region.

Cholinergic Induction of Theta-Frequency Oscillations in Hippocampal Inhibitory Interneurons and Pacing of Pyramidal Cell Firing

Cholinergic and GABAergic medial septal afferents contribute to hippocampal theta activity in part by actions on local interneurons. Interneurons near the border between stratum radiatum and stratum lacunosum-moleculare (LM) display intrinsic membrane potential oscillations at theta frequency when depolarized near threshold. First, whole-cell current-clamp recordings in rat hippocampal slices were used to examine effects of the cholinergic agonist carbachol on biocytin-labeled LM interneurons. At resting membrane potential, cells were depolarized by bath application of 25 microM carbachol, and the depolarization was sufficient to induce membrane potential oscillations (2.4 +/- 0.2 mV) that paced cell firing. Carbachol also depolarized LM interneurons in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione, (+/-)-2-amino-5-phosphonopentanoic acid, and bicuculline, indicating that cholinergic depolarization of LM cells does not depend on ionotropic glutamate or GABA(A) synaptic transmission in local circuits. Atropine blocked the depolarization, indicating that muscarinic receptors were involved. Minimal stimulation applied to visually identified LM interneurons was then used to determine if spontaneous activity in CA1 pyramidal cells can be paced by rhythmic inhibition generated by LM cells at theta frequency. Inhibitory postsynaptic potentials evoked in pyramidal cells by single minimal stimulations were followed by rebound depolarizations and action potentials. When trains of minimal stimulation were delivered, membrane potential oscillations of depolarized pyramidal cells followed the stimulation frequency. Minimal stimulation led pyramidal cell firing with an average phase of 177 degrees. Thus, muscarinic induction of theta-frequency membrane potential oscillations in LM interneurons may contribute to the generation of rhythmic inhibition that paces intrinsically generated theta activity in CA1 pyramidal cells.

Muscarinic Receptor Activity Has Multiple Effects on the Resting Membrane Potentials of CA1 Hippocampal Interneurons

Inhibitory interneurons appear to be an important target for the muscarinic actions of cholinergic inputs to the hippocampus. We investigated the effect of muscarinic receptor activity on the membrane potential (V(m)) and currents of rat hippocampal CA1 interneurons using whole-cell recording from visually identified CA1 interneurons. The predominant response observed was a muscarinic depolarization that was detected in interneurons from all layers of CA1. This depolarization was mediated by at least two mechanisms: a reduction in a potassium current and a mechanism that depended on extracellular sodium. Other interneurons responded to muscarinic agonists with a hyperpolarization or a biphasic response (hyperpolarization followed by depolarization). Hyperpolarizations and biphasic responses were found in all layers of CA1 but more frequently in stratum radiatum and stratum lacunosum moleculare. Muscarinic hyperpolarization was caused by the activation of a barium- and cesium-sensitive inwardly rectifying potassium channel. A small number of interneurons, primarily in or bordering the stratum pyramidale, produced slow membrane potential (0.04 Hz) oscillations. Many interneurons did not respond to muscarinic activity at all; half of these were in the stratum oriens. There was no strong correlation between any changes in V(m) response to muscarine and morphology, as determined by reconstruction of the interneurons. It was not possible to predict the morphology or the layer distribution of an interneuron based on the type of muscarinic membrane potential response it had. This lack of correlation between muscarinic function and morphology implies a greater complexity of interneuron function than has been realized previously.

Quantitative evidence for increase in galanin-immunoreactive terminals in the hippocampal formation following entorhinal cortex lesions in the adult rat

The projection from the entorhinal cortex to the dentate gyrus and hippocampus is severely affected in Alzheimer's disease and there is a depletion of cholinergic terminals but an upregulation of the neuropeptide galanin, which inhibits the release of acetylcholine. Evidence for changes to galanin-immunoreactive terminals in the hippocampal formation was therefore examined after unilateral entorhinal cortex lesions in the adult rat. An increase in the density of galanin-immunoreactive terminals on the lesioned side was evident in the stratum lacunosum moleculare of the hippocampus and the outer molecular layer of the dentate gyrus at 17 days post-lesion, and it increased gradually until the last time point examined, at 40 days post-lesion. Thus we demonstrate that there is an increase in galanin-immunoreactive terminals in the hippocampal formation following entorhinal cortex lesions.

Differential ontogenetic patterns of levocabastine-sensitive neurotensin NT 2 receptors and of NT 1 receptors in the rat brain revealed by in situ hybridization

The postnatal ontogeny of the levocabastine-sensitive neurotensin receptor (NT 2) mRNA was studied by in situ hybridization in the rat brain and compared with the distribution of the levocabastine-insensitive NT 1 receptor. NT 2 receptor mRNA was absent at birth from all brain structures except the ependymal cell layer lining the ventricles. The development of NT 2 receptor mRNA followed three ontogenetic patterns. The first pattern, involving the majority of the cerebral gray matter, was characterized by a continuous increase from postnatal day 5 (P5) to P30. The second one, involving regions rich in myelinated fibers such as the corpus callosum and lacunosum moleculare layer of the hippocampus, exhibited a pronounced increase between P5 and P10, peaked at P15 and was followed by a plateau or a slight decrease. The third pattern was observed in the ependymal cell layer lining the olfactory and lateral ventricles, where the high labeling already present at birth continued to increase during development. These different developmental patterns could reflect the variety of cells expressing NT 2 receptor mRNA, including neurons, protoplasmic astrocytes in gray matter, fibrous astrocytes present in myelinated fibers tracts, and ependymal cells. In contrast, NT 1 receptor mRNA, which seems to be associated only with neurons, was highly and transiently expressed during the perinatal period in the cerebral cortex, hippocampus and striatal neuroepithelium. Other regions, notably the ventral tegmental area and substantia nigra compacta, exhibited a gradual increase in NT 1 receptor signal, reaching adult levels by P21. Both the differential localization and ontogenetic profiles of NT 1 and NT 2 receptor mRNAs suggest different involvement of these two receptors in brain functions and development.

Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity

The expression of excitatory amino acid transporters (EAATs) in rat hippocampus was studied following kainic acid-induced seizure activity in vivo and in hippocampal slice cultures. Protein and mRNA levels of the glial (EAAT2) and neuronal (EAAT3) transporters were determined with affinity-purified antibodies and oligonucleotide probes, respectively. Kainate treatment decreased EAAT3 immunoreactivity in stratum lacunosum moleculare within 4 h of seizure onset. Upon pyramidal cell death (5 days after kainate treatment), EAAT3 immunoreactivity in stratum pyramidale of CA1 and in stratum lacunosum moleculare was almost completely eliminated. The rapid effect of kainate on EAAT3 expression was confirmed by in situ hybridization; EAAT3 mRNA levels were decreased in CA1 and CA3 regions within 4–8 h of seizure onset. Kainate treatment had an opposite effect on levels and expression of EAAT2. Developmental studies indicated that the rapid regulation of transporter expression was not observed in rats younger than 21 days, an observation congruent with previous reports regarding the resistance of young rats to kainate. In hippocampal organotypic cultures, which lack a major excitatory input from the entorhinal cortex, kainate produced a slow decrease in [ 3 H ] d-aspartate uptake. This study indicates that an early effect of kainate treatment consists of down-regulation of the neuronal transporter EAAT3 in restricted hippocampal regions, together with a modest increase in the expression of the glial transporter EAAT2. Differential regulation of neuronal and glial glutamate transporters may thus play a role in kainate-induced seizure, neurotoxicity and neuronal plasticity.