Roland A. Bender

Dormant basket cell hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat.

The “dormant basket cell” hypothesis suggests that postinjury hippocampal network hyperexcitability results from the loss of vulnerable neurons that normally excite insult‐resistant inhibitory basket cells. We have reexamined the experimental basis of this hypothesis in light of reports that excitatory hilar mossy cells are not consistently vulnerable and inhibitory basket cells are not consistently seizure resistant. Prolonged afferent stimulation that reliably evoked granule cell discharges always produced extensive hilar neuron degeneration and immediate granule cell disinhibition. Conversely, kainic acid‐induced status epilepticus in chronically implanted animals produced similarly extensive hilar cell loss and immediate granule cell disinhibition, but only when granule cells discharged continuously during status epilepticus. In both preparations, electron microscopy revealed degeneration of presynaptic terminals forming asymmetrical synapses in the mossy cell target zone, including some terminating on γ‐aminobutyric acid‐immunoreactive elements, but no evidence of axosomatic or axoaxonic degeneration in the adjacent granule cell layer. Although parvalbumin immunocytochemistry and in situ hybridization revealed decreased staining, this apparently was due to altered parvalbumin expression rather than basket cell death, because substance P receptor‐positive interneurons, some of which contained residual parvalbumin immunoreactivity, survived. These results confirm the inherent vulnerability of dendritically projecting hilar mossy cells and interneurons and the relative resistance of dentate inhibitory basket and chandelier cells that target granule cell somata. The variability of hippocampal cell loss after status epilepticus suggests that altered hippocampal structure and function cannot be assumed to cause the spontaneous seizures that develop in these animals and highlights the importance of confirming hippocampal pathology and pathophysiology in vivo in each case. J. Comp. Neurol. 459:44–76, 2003.

Long-term, progressive hippocampal cell loss and dysfunction induced by early-life administration of corticotropin-releasing hormone reproduce the effects of early-life stress

Stress early in postnatal life may result in long-term memory deficits and selective loss of hippocampal neurons. The mechanisms involved are poorly understood, but they may involve molecules and processes in the immature limbic system that are activated by stressful challenges. We report that administration of corticotropin-releasing hormone (CRH), the key limbic stress modulator, to the brains of immature rats reproduced the consequences of early-life stress, reducing memory functions throughout life. These deficits were associated with progressive loss of hippocampal CA3 neurons and chronic up-regulation of hippocampal CRH expression. Importantly, they did not require the presence of stress levels of glucocorticoids. These findings indicate a critical role for CRH in the mechanisms underlying the long-term effects of early-life stress on hippocampal integrity and function.

Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the 'irritable mossy cell' hypothesis.

1 Cytochemical and in vitro whole‐cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. 2 The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non‐GABAergic hilar cells, i.e. the mossy cells. 2 Dentate granule cells following trauma showed enhanced action potential discharges, and longer‐lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. 3 There was no post‐traumatic alteration in the perforant path‐evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. 4 The late EPSCs in granule cells from fluid percussion‐injured rats were not blocked by the NMDA receptor antagonist 2‐amino‐5‐phosphonovaleric acid (APV), but were eliminated by both the non‐NMDA glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and the AMPA receptor antagonist GYKI 53655. 5 In addition, the late EPSCs were not present in low (0·5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. 6 Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. 7 These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post‐traumatic dentate gyrus.

DIFFERENTIAL AND AGE-DEPENDENT EXPRESSION OF HYPERPOLARIZATION-ACTIVATED, CYCLIC NUCLEOTIDE-GATED CATION CHANNEL ISOFORMS 1–4 SUGGESTS EVOLVING ROLES IN THE DEVELOPING RAT HIPPOCAMPUS

Hyperpolarization-activated cation currents (I(h)) are found in several brain regions including thalamus and hippocampus. Important functions of these currents in promoting synchronized network activity and in determining neuronal membrane properties have been progressively recognized, but the molecular underpinnings of these currents are only emerging. I(h) currents are generated by hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs). These channel proteins are encoded by at least four HCN genes, that govern the kinetic and functional properties of the resulting channels. Because of the potential impact of I(h)-mediated coordinated neuronal activity on the maturation of the functional hippocampal network, this study focused on determining the expression of the four members of the HCN gene family throughout postnatal hippocampal development at both the regional and single cell level.The results of these experiments demonstrated that HCNs 1, 2 and 4 are differentially expressed in interneuronal and principal cell populations of the rat hippocampal formation. Expression profiles of each HCN isoform evolve during postnatal development, and patterns observed during early postnatal ages differ significantly from those in mature hippocampus. The onset of HCN expression in interneurons of the hippocampus proper precedes that in the dentate gyrus, suggesting that HCN-mediated pacing activity may be generated in hippocampal interneurons prior to those in the hilus. Taken together, these findings indicate an age-dependent spatiotemporal evolution of specific HCN expression in distinct hippocampal cell populations, and suggest that these channels serve differing and evolving functions in the maturation of coordinated hippocampal activity.

Aromatase Inhibition Abolishes LTP Generation in Female But Not in Male Mice

Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.

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.

Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus

The hyperpolarization‐activated current (Ih) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates Ih conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of Ih in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN‐subunit isoforms that contribute to this current, and their interplay with age‐dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. Ih amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow‐kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of Ih to cAMP, and the contribution of the slow component to the overall Ih, decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents.

Deletion of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Auxiliary Subunit TRIP8b Impairs Hippocampal Ih Localization and Function and Promotes Antidepressant Behavior in Mice

Output properties of neurons are greatly shaped by voltage-gated ion channels, whose biophysical properties and localization within axodendritic compartments serve to significantly transform the original input. The hyperpolarization-activated current, Ih, is mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and plays a fundamental role in influencing neuronal excitability by regulating both membrane potential and input resistance. In neurons such as cortical and hippocampal pyramidal neurons, the subcellular localization of HCN channels plays a critical functional role, yet mechanisms controlling HCN channel trafficking are not fully understood. Because ion channel function and localization are often influenced by interacting proteins, we generated a knock-out mouse lacking the HCN channel auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Eliminating expression of TRIP8b dramatically reduced Ih expression in hippocampal pyramidal neurons. Loss of Ih-dependent membrane voltage properties was attributable to reduction of HCN channels on the neuronal surface, and there was a striking disruption of the normal expression pattern of HCN channels in pyramidal neuron dendrites. In heterologous cells and neurons, absence of TRIP8b increased HCN subunit targeting to and degradation by lysosomes. Mice lacking TRIP8b demonstrated motor learning deficits and enhanced resistance to multiple tasks of behavioral despair with high predictive validity for antidepressant efficacy. We observed similar resistance to behavioral despair in distinct mutant mice lacking HCN1 or HCN2. These data demonstrate that interaction with the auxiliary subunit TRIP8b is a major mechanism underlying proper expression of HCN channels and Ih in vivo, and suggest that targeting Ih may provide a novel approach to treatment of depression.

Postnatal Expression Pattern of HCN Channel Isoforms in Thalamic Neurons: Relationship to Maturation of Thalamocortical Oscillations

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (Ih). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and Ih properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in Ih density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the Ih activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on Ih conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in Ih density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining Ih properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

Localization of HCN1 Channels to Presynaptic Compartments: Novel Plasticity That May Contribute to Hippocampal Maturation

Increasing evidence supports roles for the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, Ih, in hippocampal maturation and specifically in the evolving changes of intrinsic properties as well as network responses of hippocampal neurons. Here, we describe a novel developmental plasticity of HCN channel expression in axonal and presynaptic compartments: HCN1 channels were localized to axon terminals of the perforant path (the major hippocampal afferent pathway) of immature rats, where they modulated synaptic efficacy. However, presynaptic expression and functions of the channels disappeared with maturation. This was a result of altered channel transport to the axons, because HCN1 mRNA and protein levels in entorhinal cortex neurons, where the perforant path axons originate, were stable through adulthood. Blocking action potential firing in vitro increased presynaptic expression of HCN1 channels in the perforant path, suggesting that network activity contributed to regulating this expression. These findings support a novel developmentally regulated axonal transport of functional ion channels and suggest a role for HCN1 channel-mediated presynaptic Ih in hippocampal maturation.