A therapeutic small molecule lead enhances γ-oscillations and improves cognition/memory in Alzheimer's disease model mice.

Brain rhythms provide the timing and concurrence of brain activity required for linking together neuronal ensembles engaged in specific tasks. In particular, the γ-oscillations (30-120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here we report on a potent brain permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a new class of therapeutics for AD. As a first in CNS pharmacotherapy, our lead candidate acts as a potent, efficacious, and selective negative allosteric modulator (NAM) of the γ-aminobutyric acid type A receptors (GABA A Rs) assembled from α1ß2δ subunits. We identified these receptors through anatomical and pharmacological means to mediate the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.

Virally-induced expression of GABAA receptor δ subunits following their pathological loss reveals their role in regulating GABAA receptor assembly.

Decreased expression of the δ subunit of the GABAA receptor (GABAAR) has been found in the dentate gyrus in several animal models of epilepsy and other disorders with increased excitability and is associated with altered modulation of tonic inhibition in dentate granule cells (GCs). In contrast, other GABAAR subunits, including α4 and γ2 subunits, are increased, but the relationship between these changes is unclear. The goals of this study were to determine if viral transfection of δ subunits in dentate GCs could increase δ subunit expression, alter expression of potentially-related GABAAR subunits, and restore more normal network excitability in the dentate gyrus in a mouse model of epilepsy. Pilocarpine-induced seizures were elicited in DOCK10-Cre mice that express Cre selectively in dentate GCs, and two weeks later the mice were injected unilaterally with a Cre-dependent δ-GABAAR viral vector. At 4-6 weeks following transfection, δ subunit immunolabeling was substantially increased in dentate GCs on the transfected side compared to the nontransfected side. Importantly, α4 and γ2 subunit labeling was downregulated on the transfected side. Electrophysiological studies revealed enhanced tonic inhibition, decreased network excitability, and increased neurosteroid sensitivity in slices from the δ subunit-transfected side compared to those from the nontransfected side of the same pilocarpine-treated animal, consistent with the formation of δ subunit-containing GABAARs. No differences were observed between sides of eYFP-transfected animals. These findings are consistent with the idea that altering expression of key subunits, such as the δ subunit, regulates GABAAR subunit assemblies, resulting in substantial effects on network excitability.

Mossy Cells in the Dorsal and Ventral Dentate Gyrus Differ in Their Patterns of Axonal Projections.

Mossy cells (MCs) of the dentate gyrus (DG) are a major group of excitatory hilar neurons that are important for regulating activity of dentate granule cells. MCs are particularly intriguing because of their extensive longitudinal connections within the DG. It has generally been assumed that MCs in the dorsal and ventral DG have similar patterns of termination in the inner one-third of the dentate molecular layer. Here, we demonstrate that axonal projections of MCs in these two regions are considerably different. MCs in dorsal and ventral regions were labeled selectively with Cre-dependent eYFP or mCherry, using two transgenic mouse lines (including both sexes) that express Cre-recombinase in MCs. At four to six weeks following unilateral labeling of MCs in the ventral DG, a dense band of fibers was present in the inner one-fourth of the molecular layer and extended bilaterally throughout the rostral-caudal extent of the DG, replicating the expected distribution of MC axons. In contrast, following labeling of MCs in the dorsal DG, the projections were more diffusely distributed. At the level of transfection, fibers were present in the inner molecular layer, but they progressively expanded into the middle molecular layer and, most ventrally, formed a distinct band in this region. Optical stimulation of these caudal fibers expressing ChR2 demonstrated robust EPSCs in ipsilateral granule cells and enhanced the effects of perforant path stimulation in the ventral DG. These findings suggest that MCs in the dorsal and ventral DG differ in the distribution of their axonal projections and possibly their function.SIGNIFICANCE STATEMENT Mossy cells (MCs), a major cell type in the hilus of the dentate gyrus (DG), are unique in providing extensive longitudinal and commissural projections throughout the DG. Although it has been assumed that all MCs have similar patterns of termination in the inner molecular layer of the DG, we discovered that the axonal projections of dorsal and ventral MCs differ. While ventral MC projections exhibit the classical pattern, with dense innervation in the inner molecular layer, dorsal MCs have a more diffuse distribution and expand into the middle molecular layer where they overlap and interact with innervation from the perforant path. These distinct locations and patterns of axonal projections suggest that dorsal and ventral MCs may have different functional roles.

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABAA receptors (GABAARs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABAAR, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABAAR could be a novel approach to treatment of hyperexcitability-related alterations in FXS.

Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus.

The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT: In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit very little tonic inhibition. In an effort to increase tonic inhibition selectively in these interneurons, we used Cre-dependent viral vectors in SOM-Cre mice to achieve interneuron-specific expression of the nonsynaptic GABAAR subunits (α6 and δ) in vivo. We show, for the first time, that such recombinant GFP-tagged GABAAR subunits are expressed robustly, assemble to form functional receptors, substantially increase tonic inhibition in SOM interneurons, and alter circuit activity within the dentate gyrus.

Do structural changes in GABA neurons give rise to the epileptic state?

Identifying the role of GABA neurons in the development of an epileptic state has been particularly difficult in acquired epilepsy, in part because of the multiple changes that occur in such conditions. Although once questioned, there is now considerable evidence for loss of GABA neurons in multiple brain regions in models of acquired epilepsy. This loss can affect several cell types, including both somatostatin- and parvalbumin-expressing interneurons, and the cell type that is most severely affected can vary among brain regions and models. Because of the diversity of GABA neurons in the hippocampus and cerebral cortex, resulting functional deficits are unlikely to be compensated fully by remaining GABA neurons of other subtypes. The fundamental importance of GABA neuron loss in epilepsy is supported by findings in genetic mouse models in which GABA neurons appear to be decreased relatively selectively, and increased seizure susceptibility and spontaneous seizures develop. Alterations in remaining GABA neurons also occur in acquired epilepsy. These include alterations in inputs or receptors that could impair function, as well as morphological reorganization of GABAergic axons and their synaptic connections. Such axonal sprouting could be compensatory if normal circuits are reestablished, but the creation of aberrant circuitry could contribute to an epileptic condition. The functional effects of GABA neuron alterations thus may include not only reductions in GABAergic inhibition but also excessive neuronal synchrony and, potentially, depolarizing GABAergic influences. The combination of GABA neuron loss and alterations in remaining GABA neurons provides likely, though still unproven, substrates for the epileptic state.

Altered localization of the δ subunit of the GABAA receptor in the thalamus of α4 subunit knockout mice.

The α4 subunit of the GABAA receptor (GABAAR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABAAR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABAAR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.

Number and regional distribution of GAD65 mRNA-expressing interneurons in the rat hippocampal formation.

In rodent models for neuropsychiatric disorders reduced number of hippocampal interneurons have been reported, but the total number of GABAergic neurons in the normal rat hippocampus is yet unknown. We used in situ hybridization method to label the 65 isoform of glutamic acid decarboxylase (GAD65) and counted the number of GAD65 mRNA-expressing neurons along the entire septo-temporal axis of the hippocampus. We found that 2/3 of the interneurons were in Ammon's horn (61,590) and 1/3 in the dentate gyrus (28,000). We observed the following numbers in Ammon's horn: CA3 area 33,400, CA2 area 4,190, CA1 area 24,000 and in the dentate gyrus: 6,000 in the molecular and 9,000 in the granule cell layers and 13,000 in the hilus. GAD65 mRNA-expressing neurons were significantly more numerous in dorsal than in ventral hippocampus. The ratio between interneurons and principal cells was lowest in the granule cell layer (0.9%) and highest in hilus (21%). In Ammon's horn this ratio was constant being 13% in CA3 and 8% in CA1-2 areas. In the entire hippocampal formation, the interneuron/principal cell ratio was 6%, with a significant difference between Ammon's horn (9.5%) and the dentate gyrus (2.8%) including the hilus. Such low ratios could suggest that even a limited loss of GABAergic neurons in the hippocampus may have a considerable functional impact.

A reorganized GABAergic circuit in a model of epilepsy: evidence from optogenetic labeling and stimulation of somatostatin interneurons.

Axonal sprouting of excitatory neurons is frequently observed in temporal lobe epilepsy, but the extent to which inhibitory interneurons undergo similar axonal reorganization remains unclear. The goal of this study was to determine whether somatostatin (SOM)-expressing neurons in stratum (s.) oriens of the hippocampus exhibit axonal sprouting beyond their normal territory and innervate granule cells of the dentate gyrus in a pilocarpine model of epilepsy. To obtain selective labeling of SOM-expressing neurons in s. oriens, a Cre recombinase-dependent construct for channelrhodopsin2 fused to enhanced yellow fluorescent protein (ChR2-eYFP) was virally delivered to this region in SOM-Cre mice. In control mice, labeled axons were restricted primarily to s. lacunosum-moleculare. However, in pilocarpine-treated animals, a rich plexus of ChR2-eYFP-labeled fibers and boutons extended into the dentate molecular layer. Electron microscopy with immunogold labeling demonstrated labeled axon terminals that formed symmetric synapses on dendritic profiles in this region, consistent with innervation of granule cells. Patterned illumination of ChR2-labeled fibers in s. lacunosum-moleculare of CA1 and the dentate molecular layer elicited GABAergic inhibitory responses in dentate granule cells in pilocarpine-treated mice but not in controls. Similar optical stimulation in the dentate hilus evoked no significant responses in granule cells of either group of mice. These findings indicate that under pathological conditions, SOM/GABAergic neurons can undergo substantial axonal reorganization beyond their normal territory and establish aberrant synaptic connections. Such reorganized circuitry could contribute to functional deficits in inhibition in epilepsy, despite the presence of numerous GABAergic terminals in the region.

Effects of climbing fiber driven inhibition on Purkinje neuron spiking.

Climbing fiber (CF) input to the cerebellum is thought to instruct associative motor memory formation through its effects on multiple sites within the cerebellar circuit. We used adeno-associated viral delivery of channelrhodopsin-2 (ChR2) to inferior olivary neurons to selectively express ChR2 in CFs, achieving nearly complete transfection of CFs in the caudal cerebellar lobules of rats. As expected, optical stimulation of ChR2-expressing CFs generates complex spike responses in individual Purkinje neurons (PNs); in addition we found that such stimulation recruits a network of inhibitory interneurons in the molecular layer. This CF-driven disynaptic inhibition prolongs the postcomplex spike pause observed when spontaneously firing PNs receive direct CF input; such inhibition also elicits pauses in spontaneously firing PNs not receiving direct CF input. Baseline firing rates of PNs are strongly suppressed by low-frequency (2 Hz) stimulation of CFs, and this suppression is partly relieved by blocking synaptic inhibition. We conclude that CF-driven, disynaptic inhibition has a major influence on PN excitability and contributes to the widely observed negative correlation between complex and simple spike rates. Because they receive input from many CFs, molecular layer interneurons are well positioned to detect the spatiotemporal patterns of CF activity believed to encode error signals. Together, our findings suggest that such inhibition may bind together groups of Purkinje neurons to provide instructive signals to downstream sites in the cerebellar circuit.

Neuroanatomical clues to altered neuronal activity in epilepsy: from ultrastructure to signaling pathways of dentate granule cells.

The dynamic aspects of epilepsy, in which seizures occur sporadically and are interspersed with periods of relatively normal brain function, present special challenges for neuroanatomical studies. Although numerous morphologic changes can be identified during the chronic period, the relationship of many of these changes to seizure generation and propagation remains unclear. Mossy fiber sprouting is an example of a frequently observed morphologic change for which a functional role in epilepsy continues to be debated. This review focuses on neuroanatomically identified changes that would support high levels of activity in reorganized mossy fibers and potentially associated granule cell activation. Early ultrastructural studies of reorganized mossy fiber terminals in human temporal lobe epilepsy tissue have identified morphologic substrates for highly efficacious excitatory connections among granule cells. If similar connections in animal models contribute to seizure activity, activation of granule cells would be expected. Increased labeling with two activity-related markers, Fos and phosphorylated extracellular signal-regulated kinase, has suggested increased activity of dentate granule cells at the time of spontaneous seizures in a mouse model of epilepsy. However, neuroanatomical support for a direct link between activation of reorganized mossy fiber terminals and increased granule cell activity remains elusive. As novel activity-related markers are developed, it may yet be possible to demonstrate such functional links and allow mapping of seizure activity throughout the brain. Relating patterns of neuronal activity during seizures to the underlying morphologic changes could provide important new insights into the basic mechanisms of epilepsy and seizure generation.

Heterogeneity of the supramammillary-hippocampal pathways: evidence for a unique GABAergic neurotransmitter phenotype and regional differences.

The supramammillary nucleus (SuM) provides substantial projections to the hippocampal formation. This hypothalamic structure is involved in the regulation of hippocampal theta rhythm and therefore the control of hippocampal-dependent cognitive functions as well as emotional behavior. A major goal of this study was to characterize the neurotransmitter identity of the SuM-hippocampal pathways. Our findings demonstrate two distinct neurochemical pathways in rat. The first pathway originates from neurons in the lateral region of the SuM and innervates the supragranular layer of the dorsal dentate gyrus and, to a much lesser extent, the ventral dentate gyrus. This pathway displays a unique dual phenotype for GABAergic and glutamatergic neurotransmission. Axon terminals contain markers of GABAergic neurotransmission, including the synthesizing enzyme of GABA, glutamate decarboxylase 65, and the vesicular GABA transporter and also a marker of glutamatergic neurotransmission, the vesicular glutamate transporter 2. The second pathway originates from neurons in the most posterior and medial part of the SuM and innervates exclusively the inner molecular layer of the ventral dentate gyrus and the CA2/CA3a pyramidal cell layer of the hippocampus. The axon terminals from the medial part of the SuM contain the vesicular glutamate transporter 2 only. These data demonstrate for the first time the heterogeneity of the SuM-hippocampal pathways, not only from an anatomical but also a neurochemical point of view. These pathways, implicated in different neuronal networks, could modulate different hippocampal activities. They are likely to be involved differently in the regulation of hippocampal theta rhythm and associated cognitive functions as well as emotional behavior.

Selective reduction of cholecystokinin-positive basket cell innervation in a model of temporal lobe epilepsy.

Perisomatic inhibition from basket cells plays an important role in regulating pyramidal cell output. Two major subclasses of CA1 basket cells can be identified based on their expression of either cholecystokinin (CCK) or parvalbumin. This study examined their fates in the mouse pilocarpine model of temporal lobe epilepsy. Overall, immunohistochemical labeling of GABAergic boutons in the pyramidal cell layer of CA1 was preserved in the mouse model. However, CCK-labeled boutons in this layer were chronically reduced, whereas parvalbumin-containing boutons were conserved. Immunohistochemistry for cannabinoid receptor 1 (CB(1)), another marker for CCK-containing basket cells, also labeled fewer boutons in pilocarpine-treated mice. Hours after status epilepticus, electron microscopy revealed dark degenerating terminals in the pyramidal cell layer with lingering CCK and CB(1) immunoreactivity. In mice with recurrent seizures, carbachol-induced enhancement of spontaneous IPSCs (sIPSCs) originating from CCK-containing basket cells was accordingly reduced in CA1 pyramidal cells. By suppressing sIPSCs from CCK-expressing basket cells, a CB(1) agonist reverted the stimulatory effects of carbachol in naive mice to levels comparable with those observed in cells from epileptic mice. The agatoxin-sensitive component of CA1 pyramidal cell sIPSCs from parvalbumin-containing interneurons was increased in pilocarpine-treated mice, and miniature IPSCs were reduced, paralleling the decrease in CCK-labeled terminals. Altogether, the findings are consistent with selective reduction in perisomatic CA1 pyramidal cell innervation from CCK-expressing basket cells in mice with spontaneous seizures and a greater reliance on persisting parvalbumin innervation. This differential alteration in inhibition may contribute to the vulnerability of the network to seizure activity.

Activation of ERK by spontaneous seizures in neural progenitors of the dentate gyrus in a mouse model of epilepsy.

Cellular changes that are associated with spontaneous seizures in temporal lobe epilepsy are not well understood but could influence ongoing epilepsy-related processes. In order to identify cell signaling events that could occur at the time of spontaneous seizures, the localization of phosphorylated extracellular signal-regulated kinase (pERK) was studied in a pilocarpine mouse model of epilepsy at very short intervals (1.5-2.5 min) after detection of a spontaneous seizure. Within the hippocampal formation, immunolabeling of pERK was evident in a subpopulation of cells in the subgranular zone (SGZ) of the dentate gyrus at these short intervals. Many of these cells had a long vertical process and resembled radial glia, while others had short processes and were oriented horizontally. Labeling with a series of developmental markers demonstrated that virtually all pERK-labeled cells were neural progenitor cells (NPCs). A high percentage ( approximately 80%) of the pERK-labeled cells was labeled with either glial fibrillary acidic protein or brain lipid binding protein, indicating that these cells were radial glia-like NPCs. A smaller percentage of labeled cells expressed NeuroD, suggesting that they were later-developing NPCs that were assuming a neuronal identity. Early expression of pERK was not detected in immature neurons. Double labeling with proliferation markers demonstrated that approximately 30% of pERK-labeled NPCs expressed Mcm2, indicating that they were actively proliferating. Furthermore, virtually all radial glia-like NPCs that were in the proliferative cycle expressed pERK. These findings suggest that spontaneous seizures and associated ERK activation could contribute to the proliferation of radial glia-like NPCs in this epilepsy model.

The alpha1 subunit of the GABA(A) receptor modulates fear learning and plasticity in the lateral amygdala.

Synaptic plasticity in the amygdala is essential for emotional learning. Fear conditioning, for example, depends on changes in excitatory transmission that occur following NMDA receptor activation and AMPA receptor modification in this region. The role of these and other glutamatergic mechanisms have been studied extensively in this circuit while relatively little is known about the contribution of inhibitory transmission. The current experiments addressed this issue by examining the role of the GABA(A) receptor subunit alpha1 in fear learning and plasticity. We first confirmed previous findings that the alpha1 subunit is highly expressed in the lateral nucleus of the amygdala. Consistent with this observation, genetic deletion of this subunit selectively enhanced plasticity in the lateral amygdala and increased auditory fear conditioning. Mice with selective deletion of alpha1 in excitatory cells did not exhibit enhanced learning. Finally, infusion of a alpha1 receptor antagonist into the lateral amygdala selectively impaired auditory fear learning. Together, these results suggest that inhibitory transmission mediated by alpha1-containing GABA(A) receptors plays a critical role in amygdala plasticity and fear learning.

Deletion of GAD67 in dopamine receptor-1 expressing cells causes specific motor deficits.

The medium spiny neurons (MSNs), which comprise the direct and indirect output pathways from the striatum, use gamma-aminobutyric acid (GABA) as their major fact-acting neurotransmitter. We generated mice carrying a conditional allele of the Gad1 gene, which encodes GAD67, one of the two enzymes responsible for GABA biosynthesis, and bred them to mice expressing Cre recombinase at the dopamine D1 receptor locus (Drd1a) to selectively reduce GABA synthesis in the direct output pathway from the striatum. We show that these mice are deficient in some types of motor skills, but normal for others, suggesting a differential role for GABA release from D1 receptor-containing neurons.

Interneurons of the dentate gyrus: an overview of cell types, terminal fields and neurochemical identity.

Interneurons of the dentate gyrus are a diverse group of neurons that use GABA as their primary neurotransmitter. Morphological studies of these neurons have been challenging since no single neuroanatomical method provides a complete view of these interneurons. However, through the integration of findings obtained from multiple methods, an interesting picture of this complex group of neurons is emerging, and this review focuses on studies in rats and mice. In situ hybridization of mRNAs for the two isoforms of the GABA synthesizing enzyme, glutamate decarboxylase (GAD65 and GAD67), demonstrates the abundance of GABA neurons in the dentate gyrus and their high concentration in the hilus and along the base of the granule cell layer. Likewise, immunohistochemical studies, particularly of GAD65, demonstrate the rich fields of GABA terminals not only around the somata of granule cells but also in the dendritic regions of the molecular layer. This broad group of GABA neurons and their terminals can be subdivided according to their morphological characteristics, including the distribution of their axonal plexus, and their neurochemical identity. Intracellular labeling of single interneurons has been instrumental in demonstrating the extensiveness of their axonal plexus and the relatively specific spatial distribution of their axonal fields. These findings have led to the broad classification of interneurons into those that terminate primarily at perisomatic regions and those that innervate the dendrites of granule cells. The interneurons also can be classified according to their neuropeptide and calcium-binding protein content. These and other molecules contribute to the rich diversity of dentate interneurons and may provide opportunities for selectively regulating specific groups of GABA neurons in the dentate gyrus in order to enhance their function or protect vulnerable neurons from damage.

Altered localization of GABA(A) receptor subunits on dentate granule cell dendrites influences tonic and phasic inhibition in a mouse model of epilepsy.

Complex changes in GABA(A) receptors (GABA(A)Rs) in animal models of temporal lobe epilepsy during the chronic period include a decrease in the delta subunit and increases in the alpha4 and gamma2 subunits in the dentate gyrus. We used postembedding immunogold labeling to determine whether the subcellular locations of these subunits were also altered in pilocarpine-treated epileptic mice, and related functional changes were identified electrophysiologically. The ultrastructural studies confirmed a decrease in delta subunit labeling at perisynaptic locations in the molecular layer of the dentate gyrus where these subunits are critical for tonic inhibition. Unexpectedly, tonic inhibition in dentate granule cells was maintained in the epileptic mice, suggesting compensation by other GABA(A)Rs. An insensitivity of the tonic current to the neurosteroid tetrahydrodeoxy-corticosterone was consistent with decreased expression of the delta subunit. In the pilocarpine-treated mice, alpha4 subunit labeling remained at perisynaptic locations, but increased gamma2 subunit labeling was also found at many perisynaptic locations on granule cell dendrites, consistent with a shift of the gamma2 subunit from synaptic to perisynaptic locations and potential partnership of the alpha4 and gamma2 subunits in the epileptic animals. The decreased gamma2 labeling near the center of synaptic contacts was paralleled by a corresponding decrease in the dendritic phasic inhibition of granule cells in the pilocarpine-treated mice. These GABA(A)R subunit changes appear to impair both tonic and phasic inhibition, particularly at granule cell dendrites, and could reduce the adaptive responses of the GABA system in temporal lobe epilepsy.

A new naturally occurring GABA(A) receptor subunit partnership with high sensitivity to ethanol.

According to the rules of GABA(A) receptor (GABA(A)R) subunit assembly, alpha4 and alpha6 subunits are considered to be the natural partners of delta subunits. These GABA(A)Rs are a preferred target of low, sobriety-impairing concentrations of ethanol. Here we demonstrate a new naturally occurring GABA(A)R subunit partnership: delta subunits of hippocampal interneurons are coexpressed and colocalized with alpha1 subunits, but not with alpha4, alpha6 or any other alpha subunits. Ethanol potentiates the tonic inhibition mediated by such native alpha1/delta GABA(A)Rs in wild-type and in alpha4 subunit-deficient (Gabra4(-/-)) mice, but not in delta subunit-deficient (Gabrd(-/-)) mice. We also ruled out any compensatory upregulation of alpha6 subunits that might have accounted for the ethanol effect in Gabra4(-/-) mice. Thus, alpha1/delta subunit assemblies represent a new neuronal GABA(A)R subunit partnership present in hippocampal interneurons, mediate tonic inhibitory currents and are highly sensitive to low concentrations of ethanol.

Hippocampal cell loss in posttraumatic human epilepsy.

PURPOSE: We performed this study to determine whether significant head trauma in human adults can result in hippocampal cell loss, particularly in hilar (polymorph) and CA3 neurons, similar to that observed in animal models of traumatic brain injury. We examined the incidence of hippocampal pathology and its relation to temporal neocortical pathology, neuronal reorganization, and other variables. METHODS: Twenty-one of 200 sequential temporal lobectomies had only trauma as a risk factor for epilepsy. Tissue specimens from temporal neocortex and hippocampus were stained with glial fibrillary acidic protein (GFAP) and hematoxylin and eosin (H&E). Eleven hippocampal specimens had additional analysis of neuronal distributions by using cresyl violet and immunolabeling of a neuron-specific nuclear protein. RESULTS: The median age at onset of trauma was 19 years, the median time between trauma and onset of seizures was 2 years, and the median epilepsy duration was 16 years. The length of the latent period was inversely related to the age at the time of trauma (r=0.75; Spearman). The neocortex showed gliosis in all specimens, with hemosiderosis (n=8) or heterotopias (n=6) in some, a distribution differing from chance (p=0.02; Fisher). Hippocampal neuronal loss was found in 94% of specimens, and all of these had cell loss in the polymorph (hilar) region of the dentate gyrus. Hilar cell loss ranged from mild, when cell loss was confined to the hilus, to severe, when cell loss extended into CA3 and CA1. Some degree of mossy fiber sprouting was found in the dentate gyrus of all 10 specimens in which it was evaluated. Granule cell dispersion (n=4) was seen only in specimens with moderate to severe neuronal loss. CONCLUSIONS: Neocortical pathology was universally present after trauma. Neuronal loss in the hilar region was the most consistent finding in the hippocampal formation, similar to that found in the fluid-percussion model of traumatic head injury. These findings support the idea that head trauma can induce hippocampal epilepsy in humans in the absence of other known risk factors.