Enhanced Ras activity in pyramidal neurons induces cellular hypertrophy and changes in afferent and intrinsic connectivity in synRas mice

Neurotrophic actions are critically controlled and transmitted to cellular responses by the small G protein Ras which is therefore essential for normal functioning and plasticity of the nervous system. The present study summarises findings of recent studies on morphological changes in the neocortex of synRas mice expressing Val12-Ha-Ras in vivo under the control of the rat synapsin I promoter. In the here reported model (introduced by Heumann et al. [J. Cell Biol. 151 (2000) 1537]), transgenic Val12-Ha-Ras expression is confined to the pyramidal cell population and starts postnatally at a time, when neurons are postmitotic and their developmental maturation has been basically completed. Expression of Val12-Ha-Ras results in a significant enlargement of pyramidal neurons. Size, complexity and spine density of dendritic trees are increased, which leads, finally, to cortical expansion. However, the main morphological design principles of 'transgenic' pyramidal cells remain preserved. In addition to somato-dendritic changes, expression of Val12-Ha-Ras in pyramidal cells induces augmented axon calibres and upregulates the establishment of efferent boutons. Despite the enlargement of cortical size, the overall density of terminals representing intra- or interhemispheric, specific and non-specific afferents is unchanged or even higher in transgenic mice suggesting a significant increase in the total afferent input to the neocortex. Although interneurons do not express the transgene and are therefore excluded from direct, intrinsic Val12-Ha-Ras effects, they respond with morphological adaptations to structural changes. Thus, dendritic arbours of interneurons are extended to follow the cortical expansion and basket cells establish a denser inhibitory innervation of 'transgenic' pyramidal cells perikarya. It is concluded that expression of Val12-Ha-Ras in pyramidal neurons results in remodelling of neocortical structuring which strongly implicates a crucial involvement of Ras in cortical plasticity.     

Expression of constitutively active p21H-rasval12 in postmitotic pyramidal neurons results in increased dendritic size and complexity

The small G protein p21Ras is a critical molecular switch for relaying neurotrophic actions and is essential for normal functioning and plasticity of the nervous system. In this study, the morphogenetic effects of p21Ras were investigated on neurons in vivo. Morphological changes of layers II/III and Vb commissural pyramidal neurons of the primary somatosensory cortex were analyzed in transgenic mice expressing permanently active p21H-RasVal12 in postmitotic neurons. Pyramidal cells were retrogradely labelled with biotinylated dextran amine and subsequently traced using Neurolucida. Compared with wild-type mice, transgenic animals showed a significant increase in the surface area and volume of basal dendrites on the proximal and intermediate segments in layers II/III and on further distal segments in layer V. In addition, the surface area and volume of the trunk and of the proximal segments of oblique branches of apical dendrites were enlarged in both layers. Sholl analyses of basal and apical dendrites showed a significant increase in dendritic complexity of layer V neurons. A positive correlation was observed between the size of the basal dendrite and the neuronal soma size in the transgenic population, indicating that growth-promoting effects of p21H-RasVal12 affect both cellular compartments in parallel. However, the dendritic surface correlated with the number of tips and dendritic stem diameter in both wild-type and transgenic populations, demonstrating that these relations represent rather conservative design principles in dendritic morphology. The data presented here suggest an important role of p21Ras-dependent signaling in the final differentiation and maintenance of dendritic morphology.     

Insulin inhibits pyramidal neurons in hippocampal slices

The effect of bombesin, applied intraventricularly in the rat, was examined with regard to the central control of behavior and arousal. Infusion of 1.0 micrograms bombesin produced stereotypic grooming activity and completely eliminated observable sleep. EEG frequency analysis confirmed the absence of sleep and demonstrated normal waking patterns during behavioral stereotypy. These results support a possible physiologic function for bombesin-like peptides in grooming and the sleep-waking cycle.     

The influence of dopamine of epileptiform burst activity in hippocampal pyramidal neurons

Dopamine (DA) application to guinea pig hippocampal CA1 neurons in vitro causes hyperpolarization of the resting potential, increase in conductance, and increase in amplitude and duration of the afterhyperpolarization (AHP). Since these changes could influence repetitive firing, we performed experiments to determine whether DA-induced effects would suppress epileptogenesis in the hippocampus. Epileptiform bursts were induced by adding penicillin (3.4 mM) to the perfusion medium. Focal application of DA (40-160 microns) onto CA1 cells (n = 15) produced a hyperpolarization averaging 4.5 mV beginning in 5-20 s and lasting up to 3 min. DA also caused an increase in the amplitude and duration of slow AHPs. The frequency of spontaneous epileptiform events however was not affected. CA3 neurons (n = 6) responded to DA application with an initial 1-3 mV depolarization beginning within 5-30 s and lasting 1-2 min. In 3 cases a small hyperpolarization lasting several minutes subsequently developed. AHP duration increased 70% and amplitude increased 35% (n = 4). Along with these membrane changes the frequency of epileptiform bursting in CA3 cells slowed for 1-3 min. We added DA (10-80 microM) to the perfusion medium to see whether a significant decrease in epileptiform burst frequency might occur in the follower CA1 region if greater numbers of pacemaker CA2 and CA3 cells were exposed to DA. Spontaneous CA1 bursting was reversibly slowed, the interburst interval became variable and increased from a mean of 4 to a mean of 5-7 s (n = 6). These results suggest that DA may play a role in decreasing the incidence or frequency of epileptogenic discharges in vivo.     

Acetylcholine induced modulation of hippocampal pyramidal neurons

Applications of acetylcholine to hippocampal slices maintained in vitro resulted in slow depolarizations and simultaneous increases in membrane resistance (R_N) in hippocampal pyramidal neurons. Increases in R_N had both voltage dependent and voltage independent components. These effects were associated with increases in cell discharge frequency, and development of spontaneous as well as synaptically and directly evoked burst discharges. The increase in R_N and burst firing lasted for hours. Muscarinic antagonists blocked these actions and in addition, produced a decrease in membrane resistance, which appeared to be due to blockade of a tonic effect of acetylcholine on postsynaptic membrane properties. These findings suggest that ACh acts as a neuromodulator in the hippocampus.     

Interconnection of subsurface cisterns in mouse hippocampal pyramidal cells

In order to characterize subsurface cisterns (SSCs) in further detail, a serial section study was applied on the hippocampal pyramidal cells in mice. SSCs appeared as stacks consisting of one to five layers of flattened cisterns and one layer of a dilated cistern. These structures were interconnected at their edges.     

Immunolocalization of BK channels in hippocampal pyramidal neurons

Neurons are highly specialized cells in which the integration and processing of electrical signals critically depends on the precise localization of ion channels. For large-conductance Ca(2+)- activated K(+) (BK) channels, targeting to presynaptic membranes in hippocampal pyramidal cells was reported; however, functional evidence also suggests a somatodendritic localization. Therefore we re-examined the subcellular distribution of BK channels in mouse hippocampus using a panel of independent antibodies in a combined approach of conventional immunocytochemistry on cultured neurons, pre- and postembedding electron microscopy and immunoprecipitation. In cultured murine hippocampal neurons, the colocalization of BK channels with both pre- and postsynaptic marker proteins was observed. Electron microscopy confirmed targeting of BK channels to axonal as well as dendritic membranes of glutamatergic synapses in hippocampus. A postsynaptic localization of BK channels was also supported by the finding that the channel coimmunoprecipitated with PSD95, a protein solely expressed in the postsynaptic compartment. These results thus demonstrate that BK channels reside in both post- and presynaptic compartments of hippocampal pyramidal neurons.     

Constitutively enhanced p21Ras activity amplifies dendritic remodeling of hippocampal neurons during physical activity

In the present study we show that overexpression of constitutively active Ras amplifies the dendritic remodeling observed when animals were allowed to be physically active. The monomeric G-protein Ras is a key molecular trigger of distinct signal transduction pathways that play an important role in proper functioning of neurons. Our previous studies on Ras-transgenic synRas mice have demonstrated a considerable impact of Ras on dendritic growth, extension and synaptic connectivity of neurons. Voluntary access to a running wheel resulted in enlargement of hippocampal pyramidal cell dendrites in wild-type mice as expected. However, constitutively elevated Ras activity further enhanced dendritic growth and branching especially of apical arbors. The resultant dendritic surface gain was paralleled by a significant increase in dendritic spine density. Since Ras is crucially involved in signaling and cascades of neurotrophins that are elevated after physical activity, these results strongly suggest an important role of Ras in dendritic dynamics during induced neuronal remodeling.     

The genetic organization of neuron number in the pyramidal cell layer of hippocampal regio superior in house mice

This report concerns variations in neuron number within the pyramidal cell layer of hippocampal regio superior in 18 inbred strains of house mice. There is a genetically associated variability in the total number of neurons in this pyramidal layer. Systematic strain variations in the orientation of the pyramidal cell layer are also present. Relations between the numbers of neurons in variousexperimenter-defined sibdivisions of regio superior were examined following statistical corrections for the variations in orientation. This led to a preliminary delineation of 4genetically-defined subdivisions of the regio superior pyramidal cell layer.     

Enhanced Ras activity preserves dendritic size and extension as well as synaptic contacts of neurons after functional deprivation in synRas mice

The monomeric GTP-binding protein p21Ras has been repeatedly implicated in neuronal stability and plastic changes of the adult nervous system. Recently, we have shown that expression of constitutively active Ras protein in transgenic synRas mice results in a significant increase in the dendritic size and complexity of differentiated pyramidal neurons as well as in increased synaptic connectivity. In the present study, we examined the organization of the vibrissae-barrel cortex in synRas mice and the effects of enhanced Ras activity on deprivation-induced dendritic reorganization after vibrissectomy. The results demonstrate a significant increase in vibrissae-barrel sizes and proportional spacing between barrels in synRas mice, suggesting that the neuronal target specificity of thalamocortical terminals is preserved. Accordingly, the arrangement of double bouquet cells at the borders of barrel columns ensuring functional distinctness is unchanged. Partial vibrissectomy is followed by significant dendritic regression of corresponding pyramidal neurons in the barrel cortex of wild-type mice, which, however, could not be observed in synRas mice. The results provide the first evidence for a role of Ras in preserving neuronal structure after functional deprivation in vivo .     

Action of vasopressin on CA1 pyramidal neurons in rat hippocampal slices

The effects of arginine⁸-vasopressin (AVP) on the excitability of 47 pyramidal cells of the CA1 region of the hippocampus were determined by using intracellular recording techniques in a submerged slice preparation. Addition of 10⁻⁶ M AVP to the bathing medium evoked an increase in spike discharge which was slow in onset and only gradually reversible. The discharge was accompanied by an increase in excitatory postsynaptic potentials without significant change of the resting input resistance. AVP-induced excitation was found in 81% of ventral and 29% of dorsal hippocampal CA1 pyramidal cells. In low Ca²⁺, high Mg²⁺ solution this excitatory action by AVP was blocked. Microiontophoretic application of AVP onto apical or basal dendrites or the cell body did not result in excitation. These observations suggest that the action of AVP on CA1 pyramidal cells is transsynaptic and is more pronounced in ventral than dorsal CA1.     

Absence of hippocampal mossy fiber sprouting in transgenic mice overexpressing brain-derived neurotrophic factor

Excess neuronal activity upregulates the expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in adult hippocampus. Nerve growth factor has been shown to contribute the induction of aberrant hippocampal mossy fiber sprouting in the inner molecular layer of the dentate gyrus, however the role of prolonged brain-derived neurotrophic factor exposure is uncertain. We examined the distribution and plasticity of mossy fibers in transgenic mice with developmental overexpression of brain-derived neurotrophic factor. Despite 2--3-fold elevated BDNF levels in the hippocampus sufficient to increase the intensity of neuropeptide Y immunoreactivity in interneurons, no visible changes in mossy fiber Timm staining patterns were observed in the inner molecular layer of adult mutant hippocampus compared to wild-type mice. In addition, no changes of the mRNA expression of two growth-associated proteins, GAP-43 and SCG-10 were found. These data suggest that early and persistent elevations of brain-derived neurotrophic factor in granule cells are not sufficient to elicit this pattern of axonal plasticity in the hippocampus.     

Axonal remodeling during postnatal maturation of CA3 hippocampal pyramidal neurons

Anatomical substrates were investigated for local circuit hyperexcitability that occurs in the CA3 subfield of the rat hippocampus during postnatal week 2. A transient excess of excitatory local circuit connectivity was hypothesized to underlie this hyperexcitability. To test this hypothesis, recurrent excitatory axon arbors from single biocytin-filled CA3 pyramidal cells were reconstructed. Arbors were analyzed in segments of area CA3 comparable in size to in vitro minislice preparations, which were shown to reproduce the developmental hyperexcitability seen in intact slices during postnatal week 2. Segments were then adjusted for hippocampal growth, based on age-dependent changes in neuron density in stratum pyramidale. Axon arbors were found to be short and possessed very few branches during the first postnatal week. By the second postnatal week, arbors had undergone dramatic growth and were much longer and more complex in their branching patterns. By adulthood, a significant decrease in all measures of arbor length and complexity was observed. Following growth adjustment, measures of axon length and varicosity number during week 2 were not significantly different from that of adulthood. However, the number of axon branches decreased by 50%. These results suggest that, during early postnatal life, there is exuberant outgrowth of local CA3 recurrent axons, and with maturation these recurrent collaterals are remodeled. Short-ranging, profusely branched axons appear to be replaced by longer-ranging arbors that possess fewer branches. Maturational changes in the dendritic location rather than the number of early-formed recurrent excitatory synapses may explain developmental hyperexcitability of the hippocampal CA3 subfield. J. Comp. Neurol. 384:165-180, 1997. © 1997 Wiley-Liss, Inc.     

Mapping the electrophysiological and morphological properties of CA1 pyramidal neurons along the longitudinal hippocampal axis

Differences in behavioral roles, anatomical connectivity, and gene expression patterns in the dorsal, intermediate, and ventral regions of the hippocampus are well characterized. Relatively fewer studies have, however, focused on comparing the physiological properties of neurons located at different dorsoventral extents of the hippocampus. Recently, we reported that dorsal CA1 neurons are less excitable than ventral neurons. There is little or no information for how neurons in the intermediate hippocampus compare to those from the dorsal and ventral ends. Also, it is not known whether the transition of properties along the dorsoventral axis is gradual or segmented. In this study, we developed a statistical model to predict the dorsoventral position of transverse hippocampal slices. Using current clamp recordings combined with this model, we found that CA1 neurons in dorsal, intermediate, and ventral hippocampus have distinct electrophysiological and morphological properties and that the transition in most (but not all) of these properties from the ventral to dorsal end is gradual. Using linear and segmented regression analyses, we found that input resistance and resting membrane potential changed linearly along the V–D axis. Interestingly, the transition in resonance frequency, rebound slope, dendritic branching in stratum radiatum, and action potential properties was segmented along the V–D axis. Together, the findings from this study highlight the heterogeneity in CA1 neuronal properties along the entire longitudinal axis of hippocampus. © 2015 Wiley Periodicals, Inc.     

Estrogen induces elevation of cAMP-dependent protein kinase activity in immortalized hippocampal neurons: imaging in living cells

Summary. 17β-estradiol-stimulated PKA activation in living differentiated H19-7 cells was visualized using DR2, a cell-permeable fluorescent PKA substrate. Estradiol induced marked changes of fluorescence in cytosolic areas and these were inhibited by a PKA inhibitor. Received October 15, 2001; accepted December 12, 2001     

Spider toxin (JSTX) blocks glutamate synapse in hippocampal pyramidal neurons

Effects of spider toxin (JSTX)--a specific blocker of glutamate receptors--on single pyramidal neurons of the hippocampus were studied using tissue slices in vitro. JSTX blocked the synaptic response in CA1 pyramidal cells evoked by Schaffer collateral stimulation without affecting the antidromic spike potential. The toxin suppressed glutamate-induced cell firings whereas it had little effect on aspartate-induced responses. The results suggest that glutamate is a neurotransmitter of the Schaffer collateral input to CA1 pyramidal neurons.     

Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons

The so-called stress response involving up-regulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia, or oxidative stress. Some members of the group of small Hsps (sHsps) seem to play a neuroprotective role in the brain. Here we analyzed the expression of all 11 sHsps in the rat brain by using RNA in situ hybridization and quantitative real-time RT-PCR. Additionally, we investigated sHsps in cultured neurons exposed to heat shock. We found seven sHsps to be expressed in the rat brain, with HspB5 (αB-crystallin), HspB6 (Hsp20), and HspB11 (Hsp16.2) showing the highest expression levels (4-24% of reference genes) followed by HspB1 (Hsp25) and HspB8 (Hsp22; 0.1-2% of reference genes), all being widely expressed in the brain areas investigated. HspB2 (MKBP) and HspB3, however, showed selective expression in only some regions (B2: cortex and hippocampus, B3: cortex and cerebellum). Whereas HspB5 was expressed mainly in the white matter, HspB6 showed the greatest expression in the cerebellar cortex, and HspB11 was widely distributed over the whole brain. In cultured hippocampal neurons, heat shock led to an increase of HspB1 and HspB8 mRNA and additionally HspB5 protein. Our data indicate that the sHsps induced by heat shock, HspB1, B5, and B8, might be especially involved in neuroprotection under stress conditions. The other sHsps showing constant neuronal expression may play a constitutive role or may be up-regulated and important in types of stresses other than heat shock.     

Postnatal nectin‐3 knockdown induces structural abnormalities of hippocampal principal neurons and memory deficits in adult mice

The early postnatal stage is a critical period of hippocampal neurodevelopment and also a period of high vulnerability to adverse life experiences. Recent evidence suggests that nectin‐3, a cell adhesion molecule, mediates memory dysfunction and dendritic alterations in the adult hippocampus induced by postnatal stress. But it is unknown whether postnatal nectin‐3 reduction alone is sufficient to alter hippocampal structure and function in adulthood. Here, we down regulated hippocampal expression of nectin‐3 and its heterophilic adhesion partner nectin‐1, respectively, from early postnatal stage by injecting adeno‐associated virus (AAV) into the cerebral lateral ventricles of neonatal mice (postnatal day 2). We found that suppression of nectin‐3, but not nectin‐1, expression from the early postnatal stage impaired hippocampus‐dependent novel object recognition and spatial object recognition in adult mice. Moreover, AAV‐mediated nectin‐3 knockdown significantly reduced dendritic complexity and spine density of pyramidal neurons throughout the hippocampus, whereas nectin‐1 knockdown only induced the loss of stubby spines in CA3. Our data provide direct evidence that nectins, especially nectin‐3, are necessary for postnatal hippocampal development of memory functions and structural integrity.     

5-HT4 receptor activation induces long-lasting EPSP-spike potentiation in CA1 pyramidal neurons

Recent studies implicated involvement of the 5-hydroxytryptamine4 (5-HT4) receptor in cognitive and emotional processes. The highest 5-HT4 receptor densities in the brain are found in the limbic system including the hippocampus. Here we used the selective 5-HT4 receptor full agonist, N-pentyl-N'-aminoguanidine carbazimidamide (SDZ-216454) to characterize effects of 5-HT4 receptor activation in whole-cell and field recordings in the area CA1 in hippocampal slices prepared from 3 to 4- and 6 to 9-week-old rats, respectively. Extracellular recordings showed that transient 5-HT4 receptor activation by 10-20 min application of SDZ-216454 induces field excitatory postsynaptic potential (fEPSP)-population spike potentiation (ESP(5-HT4)), which persisted for as long as we held the recordings (> 2 h). ESP(5-HT4) displayed characteristics different from EPSP-spike potentiation that accompanies long-term potentiation; it developed without an associated increase in synaptic transmission, was independent on afferent input, activity of postsynaptic neurons and N-methyl-d-aspartate receptor activation; and was expressed in the presence of GABA receptor antagonists. ESP(5-HT4) was also induced by transient application of the natural neurotransmitter, 5-HT. The increase in the evoked population spike (PS) induced by SDZ-216454 was not prevented by blockers of hyperpolarization-activated cation current (Ih), Cs+ and ZD-7288, but was mimicked and occluded by 150 microm Ba2+. Whole-cell voltage-clamp recordings from pyramidal neurons demonstrated that SDZ-216454 application increases membrane resistance with a concomitant decrease in a Ba2+-sensitive inwardly rectifying K+ current and the Ba2+-insensitive K+ current underlying slow afterhyperpolarization (I(sAHP)). We conclude that 5-HT4 receptor activation may cause a long-lasting excitability increase in CA1 pyramidal neurons by inhibition of a Ba2+-sensitive inwardly rectifying K+ current.     

Focal and generalized seizure activity after local hippocampal or cortical ablation of NaV1.1 channels in mice

Early onset seizures are a hallmark of Dravet syndrome. Previous studies in rodent models have shown that the epileptic phenotype is caused by loss-of-function of voltage-gated Na_V1.1 sodium channels, which are chiefly expressed in γ-aminobutyric acid (GABA)ergic neurons. Recently, a possibly critical role has been attributed to the hippocampus in the seizure phenotype, as local hippocampal ablation of Na_V1.1 channels decreased the threshold for hyperthermia-induced seizures. However, the effect of ablation of Na_V1.1 channels restricted to cortical sites has not been tested. Here we studied local field potential (LFP) and behavior in mice following local hippocampal and cortical ablation of Scn1a, a gene encoding the α1 subunit of Na_V1.1 channels, and we compared seizure characteristics with those of heterozygous global knockout Scn1^-/+ mice. We found a high incidence of spontaneous seizures following either local hippocampal or cortical ablation, notably during a transient time window, similar to Scn1a^-/+ mice. Nonconvulsive seizure activity in the injected area was common and preceded generalized seizures. Moreover, mice were susceptible to hyperthermia-induced seizures. In conclusion, local ablation of Na_V1.1 channels in the hippocampus and cortex results in focal seizure activity that can generalize. These data indicate that spontaneous epileptic activity may initiate in multiple brain regions in Dravet syndrome.