Showing papers on "GABAergic published in 2003"

GABAergic dysfunction in mood disorders

Abstract: The authors review the available literature on the preclinical and clinical studies involving GABAergic neurotransmission in mood disorders. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter present almost exclusively in the central nervous system (CNS), distributed across almost all brain regions, and expressed in interneurons modulating local circuits. The role of GABAergic dysfunction in mood disorders was first proposed 20 years ago. Preclinical studies have suggested that GABA levels may be decreased in animal models of depression, and clinical studies reported low plasma and CSF GABA levels in mood disorder patients. Also, antidepressants, mood stabilizers, electroconvulsive therapy, and GABA agonists have been shown to reverse the depression-like behavior in animal models and to be effective in unipolar and bipolar patients by increasing brain GABAergic activity. The hypothesis of reduced GABAergic activity in mood disorders may complement the monoaminergic and serotonergic theories, proposing that the balance between multiple neurotransmitter systems may be altered in these disorders. However, low GABAergic cortical function may probably be a feature of a subset of mood disorder patients, representing a genetic susceptibility. In this paper, we discuss the status of GABAergic hypothesis of mood disorders and suggest possible directions for future preclinical and clinical research in this area.

GABA, γ-hydroxybutyric acid, and neurological disease

Abstract: γ-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. GABA is converted from glutamic acid by the action of glutamic acid decarboxylase (GAD) of which two isoforms exist GAD65 and GAD67. GABA then is broken down, both within the cell and in the synaptic cleft by GABA transaminase to form succinic semialdehyde. In turn, succinic semialdehyde is converted either to succinic acid by succinic semialdehyde dehydrogenase or into γ-hydroxybutyric acid (GHB) by succinic semialdehyde reductase. Because GABA modulates the majority of inhibition that is ongoing in the brain, perturbations in GABAergic inhibition have the potential to result in seizures. Therefore, the most common disorder in which GABA is targeted as a treatment is epilepsy. However, other disorders such as psychiatric disease, spasticity, and stiff-person syndrome all have been related to disorders of GABAergic function in the brain. This review covers the roles of GABAergic neurotransmission in epilepsy, anxiety disorders, schizophrenia, stiff-person syndrome, and premenstrual dysphoric disorder. In the final section of this review, the GABA metabolite GHB is discussed in terms of its physiological significance and its role in epilepsy, sleep disorders, drug and alcohol addiction, and an inborn error of GABA metabolism, succinic semialdehyde dehydrogenase deficiency. Ann Neurol 2003;54 (suppl 6):S3–S12

Increased cortical GABA concentrations in depressed patients receiving ECT.

Abstract: OBJECTIVE: Reduced γ-aminobutyric acid (GABA) concentrations have been reported in the plasma, CSF, and cortex of depressed subjects. Of interest is that ECT, one of the most effective treatments for severe refractory depression, produces considerable anticonvulsant effects that may be related to increased GABAergic transmission. The purpose of this study was to determine if cortical GABA concentrations increase following a course of ECT. METHOD: Occipital cortex GABA concentrations in eight depressed patients were measured by using proton magnetic resonance spectroscopy before and after a course of ECT. RESULTS: A significant increase in occipital cortex GABA concentrations was seen following ECT treatment of depression. CONCLUSIONS: Occipital cortex GABA concentrations increase two-fold following ECT. This suggests possible GABAergic involvement in ECT’s mechanism of anticonvulsant and antidepressant actions.

Reduced Inhibition of Dentate Granule Cells in a Model of Temporal Lobe Epilepsy

Abstract: Patients and models of temporal lobe epilepsy have fewer inhibitory interneurons in the dentate gyrus than controls, but it is unclear whether granule cell inhibition is reduced. We report the loss of GABAergic inhibition of granule cells in the temporal dentate gyrus of pilocarpine-induced epileptic rats. In situ hybridization for GAD65 mRNA and immunocytochemistry for parvalbumin and somatostatin confirmed the loss of inhibitory interneurons. In epileptic rats, granule cells had prolonged EPSPs, and they discharged more action potentials than controls. Although the conductances of evoked IPSPs recorded in normal ACSF were not significantly reduced and paired-pulse responses showed enhanced inhibition of granule cells from epileptic rats, more direct measures of granule cell inhibition revealed significant deficiencies. In granule cells from epileptic rats, evoked monosynaptic IPSP conductances were <40% of controls, and the frequency of GABA(A) receptor-mediated spontaneous and miniature IPSCs (mIPSCs) was <50% of controls. Within 3-7 d after pilocarpine-induced status epilepticus, miniature IPSC frequency had decreased, and it remained low, without functional evidence of compensatory synaptogenesis by GABAergic axons in chronically epileptic rats. Both parvalbumin- and somatostatin-immunoreactive interneuron numbers and the frequency of both fast- and slow-rising GABA(A) receptor-mediated mIPSCs were reduced, suggesting that loss of inhibitory synaptic input to granule cells occurred at both proximal/somatic and distal/dendritic sites. Reduced granule cell inhibition in the temporal dentate gyrus preceded the onset of spontaneous recurrent seizures by days to weeks, so it may contribute, but is insufficient, to cause epilepsy.

Genetic disruption of cortical interneuron development causes region- and GABA cell type-specific deficits, epilepsy, and behavioral dysfunction.

Abstract: The generation of properly functioning circuits during brain development requires precise timing of cell migration and differentiation Disruptions in the developmental plan may lead to neurological and psychiatric disorders Neocortical circuits rely on inhibitory GABAergic interneurons, the majority of which migrate from subcortical sources We have shown that the pleiotropic molecule hepatocyte growth factor/scatter factor (HGF/SF) mediates interneuron migration Mice with a targeted mutation of the gene encoding urokinase plasminogen activator receptor (uPAR), a key component in HGF/SF activation and function, have decreased levels of HGF/SF and a 50% reduction in neocortical GABAergic interneurons at embryonic and perinatal ages Disruption of interneuron development leads to early lethality in most models Thus, the long-term consequences of such perturbations are unknown Mice of the uPAR −/− strain survive until adulthood, and behavior testing demonstrates that they have an increased anxiety state The uPAR −/− strain also exhibits spontaneous seizure activity and higher susceptibility to pharmacologically induced convulsions The neocortex of the adult uPAR −/− mouse exhibits a dramatic region- and subtype-specific decrease in GABA-immunoreactive interneurons Anterior cingulate and parietal cortical areas contain 50% fewer GABAergic interneurons compared with wild-type littermates However, interneuron numbers in piriform and visual cortical areas do not differ from those of normal mice Characterization of interneuron subpopulations reveals a near complete loss of the parvalbumin subtype, with other subclasses remaining intact These data demonstrate that a single gene mutation can selectively alter the development of cortical interneurons in a region- and cell subtype-specific manner, with deficits leading to long-lasting changes in circuit organization and behavior

Identified sources and targets of slow inhibition in the neocortex.

Abstract: There are two types of inhibitory postsynaptic potentials in the cerebral cortex. Fast inhibition is mediated by ionotropic gamma-aminobutyric acid type A (GABA(A)) receptors, and slow inhibition is due to metabotropic GABA(B) receptors. Several neuron classes elicit inhibitory postsynaptic potentials through GABA(A) receptors, but possible distinct sources of slow inhibition remain unknown. We identified a class of GABAergic interneurons, the neurogliaform cells, that, in contrast to other GABA-releasing cells, elicited combined GABA(A) and GABA(B) receptor-mediated responses with single action potentials and that predominantly targeted the dendritic spines of pyramidal neurons. Slow inhibition evoked by a distinct interneuron in spatially restricted postsynaptic compartments could locally and selectively modulate cortical excitability.

BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl- co-transporter KCC2.

Abstract: Spontaneous neural activity is a basic property of the developing brain, which regulates key developmental processes, including migration, neural differentiation and formation and refinement of connections The mechanisms regulating spontaneous activity are not known By using transgenic embryos that overexpress BDNF under the control of the nestin promoter, we show here that BDNF controls the emergence and robustness of spontaneous activity in embryonic hippocampal slices Further, BDNF dramatically increases spontaneous co-active network activity, which is believed to synchronize gene expression and synaptogenesis in vast numbers of neurons In fact, BDNF raises the spontaneous activity of E18 hippocampal neurons to levels that are typical of postnatal slices We also show that BDNF overexpression increases the number of synapses at much earlier stages (E18) than those reported previously Most of these synapses were GABAergic, and GABAergic interneurons showed hypertrophy and a 3-fold increase in GAD expression Interestingly, whereas BDNF does not alter the expression of GABA and glutamate ionotropic receptors, it does raise the expression of the recently cloned K(+)/Cl(-) KCC2 co-transporter, which is responsible for the conversion of GABA responses from depolarizing to inhibitory, through the control of the Cl(-) potential Together, results indicate that both the presynaptic and postsynaptic machineries of GABAergic circuits may be essential targets of BDNF actions to control spontaneous activity The data indicate that BDNF is a potent regulator of spontaneous activity and co-active networks, which is a new level of regulation of neurotrophins Given that BDNF itself is regulated by neuronal activity, we suggest that BDNF acts as a homeostatic factor controlling the emergence, complexity and networking properties of spontaneous networks

Distinct electrophysiological properties of glutamatergic, cholinergic and GABAergic rat septohippocampal neurons: novel implications for hippocampal rhythmicity.

Abstract: The medial septum-diagonal band complex (MSDB) contains cholinergic and non-cholinergic neurons known to play key roles in learning and memory processing, and in the generation of hippocampal theta rhythm. Electrophysiologically, several classes of neurons have been described in the MSDB, but their chemical identity remains to be fully established. By combining electrophysiology with single-cell RT-PCR, we have identified four classes of neurons in the MSDB in vitro. The first class displayed slow-firing and little or no Ih, and expressed choline acetyl-transferase mRNA (ChAT). The second class was fast-firing, had a substantial Ih and expressed glutamic acid decarboxylase 67 mRNA (GAD67), sometimes co-localized with ChAT mRNAs. A third class exhibited fast- and burst-firing, had an important Ih and expressed GAD67 mRNA also occasionally co-localized with ChAT mRNAs. The ionic mechanism underlying the bursts involved a low-threshold spike and a prominent Ih current, conductances often associated with pacemaker activity. Interestingly, we identified a fourth class that expressed transcripts solely for one or two of the vesicular glutamate transporters (VGLUT1 and VGLUT2), but not ChAT or GAD. Some putative glutamatergic neurons displayed electrophysiological properties similar to ChAT-positive slow-firing neurons such as the occurrence of a very small Ih, but nearly half of glutamatergic neurons exhibited cluster firing with intrinsically generated voltage-dependent subthreshold membrane oscillations. Neurons belonging to each of the four described classes were found among septohippocampal neurons by retrograde labelling. We provide results suggesting that slow-firing cholinergic, fast-firing and burst-firing GABAergic, and cluster-firing glutamatergic neurons, may each uniquely contribute to hippocampal rhythmicity in vivo.

Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles

Abstract: l-Glutamic acid decarboxylase (GAD) exists as both membrane-associated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of γ-aminobutyric acid (GABA) by membrane-associated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [3H]GABA from [3H]l-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated Ca2+/calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.

Hypothalamic regulation of sleep and arousal.

Abstract: The hypnogenic function of the rostral hypothalamic region, particularly the preoptic area (POA) was established previously on the basis of lesion, neuronal unit recording, and neurochemical and thermal stimulation studies. Recent studies have mapped the locations of putative sleep-promoting neurons in the POA using c-Fos immunostaining techniques and confirmed these findings with electrophysiological methods. Segregated groups of sleep-active neurons have been localized in the ventrolateral POA (vlPOA) and median preoptic nucleus (MnPN). MnPN and vlPOA sleep-active neurons express the inhibitory transmitter, GABA. In vlPOA neurons, GABA is co-localized with a second inhibitory transmitter, galanin. Descending projections from these sites terminate in putative arousal-promoting cell groups, including histaminergic, serotonergic, orexinergic, noradrenergic, and cholinergic neurons. These findings suggest the hypothesis that non-REM sleep occurs as a consequence of GABAergic and galaninergic inhibition of arousal-promoting neurons resulting from activation of vlPOA and MnPN sleep-promoting neurons. In support of this hypothesis, it was shown that putative sleep-promoting and arousal-promoting neurons exhibit reciprocal changes in discharge across the sleep-wake cycle and that GABA release in wake-promoting sites increases during nonREM sleep. In addition, some POA sleep-active neurons are warm-sensitive. Local POA warming inhibits discharge of multiple arousal-promoting neuronal groups. POA warming, unit recording, and lesion studies also show that POA regulates the amount of delta EEG activity within nonREM sleep, and index of the depth of sleep. Finally, there is evidence that arousal systems inhibit vlPOA and MnPN neurons and the POA hypnogenic mechanism. Mutually-inhibitory interactions between sleep-promoting and arousal-promoting systems are hypothesized to form a functional sleep-wake switch.

Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain

Abstract: GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABA(A) and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA-immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA- and/or glycine-immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham-operated and nai;ve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I-III of the ipsilateral dorsal horn that showed GABA- or glycine-immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham-operated or nai;ve animals. In addition, we did not see any evidence for alterations of GABA- or glycine-immunostaining in the neuropil of laminae I-III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

Subcellular localization of metabotropic GABA(B) receptor subunits GABA(B1a/b) and GABA(B2) in the rat hippocampus.

Abstract: Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically. Using preembedding immunohistochemical methods combined with quantitative analysis of GABAB receptor subunit immunoreactivity, this study provides a detailed description of the cellular and subcellular localization of GABAB1a/b and GABAB2 in the rat hippocampus. At the light microscopic level, an overlapping distribution of GABAB1a/b and GABAB2 was revealed in the dendritic layers of the hippocampus. In addition, expression of the GABAB1a/b subunit was found in somata of CA1 pyramidal cells and of a subset of GABAergic interneurons. At the electron microscopic level, immunoreactivity for both subunits was observed on presynaptic and, more abundantly, on postsynaptic elements. Presynaptically, subunits were mainly detected in the extrasynaptic membrane and occasionally over the presynaptic membrane specialization of putative glutamatergic and, to a lesser extent, GABAergic axon terminals. Postsynaptically, the majority of GABAB receptor subunits were localized to the extrasynaptic plasma membrane of spines and dendritic shafts of principal cells and shafts of interneuron dendrites. Quantitative analysis revealed enrichment of GABAB1a/b around putative glutamatergic synapses on spines and an even distribution on dendritic shafts of pyramidal cells contacted by GABAergic boutons. The association of GABAB receptors with glutamatergic synapses at both presynaptic and postsynaptic sides indicates their intimate involvement in the modulation of glutamatergic neurotransmission. The dominant extrasynaptic localization of GABAB receptor subunits suggests that their activation is dependent on spillover of GABA requiring simultaneous activity of populations of GABAergic cells as it occurs during population oscillations or epileptic seizures.

Neuronal Glutamate Uptake Contributes to GABA Synthesis and Inhibitory Synaptic Strength

Abstract: Neurons must maintain a supply of neurotransmitter in their presynaptic terminals to fill synaptic vesicles. GABA is taken up into inhibitory terminals by transporters or is synthesized from glutamate by glutamic acid decarboxylase. Here we report that glutamate transporters supply GABAergic terminals in the hippocampus with glutamate, which is then used to synthesize GABA for filling synaptic vesicles. Glutamate transporter antagonists reduced IPSC and miniature IPSC (mIPSC) amplitudes, consistent with a reduction in the amount of GABA packaged into each synaptic vesicle. This reduction occurred rapidly and independently of synaptic activity, suggesting that modulation of vesicular GABA content does not require vesicle release and refilling. Raising extracellular glutamate levels increased mIPSC amplitudes by enhancing glutamate uptake and, consequently, GABA synthesis. These results indicate that neuronal glutamate transporters strengthen inhibitory synapses in response to extracellular glutamate. This modulation appears to occur under normal conditions and may constitute a negative feedback mechanism to combat hyperexcitability.

Loss of interneurons innervating pyramidal cell dendrites and axon initial segments in the CA1 region of the hippocampus following pilocarpine-induced seizures.

Abstract: In the pilocarpine model of chronic limbic seizures, vulnerability of GABAergic interneurons to excitotoxic damage has been reported in the hippocampal CA1 region. However, little is known about the specific types of interneurons that degenerate in this region. In order to characterize these interneurons, we performed quantitative analyses of the different populations of GABAergic neurons labeled for their peptide or calcium-binding protein content. Our data demonstrate that the decrease in the number of GAD mRNA-containing neurons in the stratum oriens of CA1 in pilocarpine-treated rats involved two subpopulations of GABAergic interneurons: interneurons labeled for somatostatin only (O-LM and bistratified cells) and interneurons labeled for parvalbumin only (basket and axo-axonic cells). Stratum oriens interneurons labeled for somatostatin/calbindin or somatostatin/parvalbumin were preserved. The decrease in number of somatostatin- and parvalbumin-containing neurons was observed as early as 72 hours after the sustained seizures induced by pilocarpine injection. Many degenerating cell bodies in the stratum oriens and degenerating axon terminals in the stratum lacunosum-moleculare were observed at 1 and 2 weeks after injection. In addition, the synaptic coverage of the axon initial segment of CA1 pyramidal cells was significantly decreased in pilocarpine-treated animals. These results indicate that the loss of somatostatin-containing neurons corresponds preferentially to the degeneration of interneurons with an axon projecting to stratum lacunosum-moleculare (O-LM cells) and suggest that the death of these neurons is mainly responsible for the deficit of dendritic inhibition reported in this region. We demonstrate that the loss of parvalbumin-containing neurons corresponds to the death of axo-axonic cells, suggesting that perisomatic inhibition and mechanisms controlling action potential generation are also impaired in this model.

Localization of the GABAergic and non-GABAergic neurons projecting to the sublaterodorsal nucleus and potentially gating paradoxical sleep onset.

Abstract: We recently determined in rats that iontophoretic application of bicuculline or gabazine [two GABAa antagonists] and kainic acid (a glutamate agonist) in the sublaterodorsal nucleus (SLD) induces with a very short latency a paradoxical sleep-like state. From these results, we proposed that GABAergic and glutamatergic inputs to the SLD paradoxical sleep (PS)-executive neurons gate the onset of PS [R. Boissard et al. (2002) Eur. J. Neurosci., 16, 1959-1973]. We therefore decided to determine the origin of the GABAergic and non-GABAergic inputs to the SLD combining ejection of a retrograde tracer [cholera-toxin B subunit (CTb)] with glutamate decarboxylase (GAD) immunohistochemistry. The presence of GAD-immunoreactive neurons in the SLD was confirmed. Then, following CTb ejections centred on the SLD, combined with GAD and CTb immunohistochemistry, double-labelled cells were observed in the mesencephalic and pontine reticular nuclei and to a lesser extent the parvicellular reticular nucleus. A large number of GAD-negative retrogradely labelled cells was also seen in these structures as well as in the primary motor area of the frontal cortex, the central nucleus of the amygdala, the ventral and lateral bed nucleus of the stria terminalis, the lateral hypothalamic area, the lateral and ventrolateral periaqueductal grey and the lateral paragigantocellular reticular nucleus. From these results, we propose that the activation of PS-executive neurons from the SLD is due to the removal of a tonic inhibition from GABAergic neurons localized in the SLD, and the mesencephalic and pontine reticular nuclei. Strong non-GABAergic inputs to the SLD could be excitatory and responsible for the tonic glutamatergic input on the PS-on neurons we have previously described. They could also terminate on SLD GABAergic interneurons and be indirectly responsible for the inhibition of the PS-on neurons during waking and slow-wave sleep.

Brain-Derived Neurotrophic Factor Modulation of GABAergic Synapses by Postsynaptic Regulation of Chloride Transport

Abstract: Brain-derived neurotrophic factor (BDNF) potentiates excitatory synapses in a variety of systems by promoting presynaptic transmitter release. The existing evidence indicates that BDNF attenuates inhibitory transmission, but reports differ considerably in their characterization of the effect and proposed mechanisms. We examined the effects of exogenously applied BDNF on EPSCs and IPSCs recorded from functionally identified neurons in dissociated rat hippocampal cultures. When recording from glutamatergic neurons, we found that BDNF exerted differential effects at excitatory versus inhibitory synapses: increasing amplitude of EPSCs but slightly decreasing that of IPSCs. Furthermore, when recording from GABAergic neurons, we found that BDNF increased the IPSC amplitude. That these differential BDNF effects reflect distinct presynaptic and postsynaptic mechanisms was suggested by the BDNF-induced changes in miniature EPSCs and IPSCs. An increased mini-frequency was found at all synapses, indicating elevated presynaptic transmitter secretion; a change in the amplitude of mini-IPSCs was found at GABAergic cells, suggesting postsynaptic modulation of GABA responses. Selective postsynaptic mechanisms were further examined by comparing the effect of BDNF on GABA-induced currents recorded from glutamatergic versus GABAergic cells. For GABAergic but not glutamatergic postsynaptic cells, BDNF induced a shift in the reversal potential (EIPSC) toward more positive levels, hence reducing the inhibitory action of IPSCs. This BDNF-induced effect correlates with the existing level of furosemide-sensitive K+-Cl- transport activity in the postsynaptic cell. Thus, BDNF may decrease the efficacy of inhibitory transmission by acute postsynaptic downregulation of Cl- transport, in addition to its well known presynaptic effect.

Parvalbumin, calbindin, or calretinin in cortically projecting and GABAergic, cholinergic, or glutamatergic basal forebrain neurons of the rat.

Abstract: The basal forebrain (BF) plays an important role in modulating cortical activity and facilitating processes of attention, learning, and memory. This role is subserved by cholinergic neurons but also requires the participation of other noncholinergic neurons. Noncholinergic neurons include -amino butyric acidergic (GABAergic) neurons, some of which project in parallel with the cholinergic cells to the cerebral cortex, others of which project caudally or locally. With the original aim of distinguishing different subgroups of GABAergic neurons, we examined immunostaining for the calcium binding proteins (CBPs) parvalbumin (Parv), calbindin (Calb), and calretinin (Calret) in the rat. Although the CBP cell groups were distributed in a coextensive manner with the GABAergic cells, they were collectively more numerous. Of cells retrogradely labeled with cholera toxin (CT) from the prefrontal or parietal cortex, Parv and Calb cells, but not Calret cells, represented substantial proportions (35–45% each) that collectively were greater than that of GABAergic projection neurons. From dual immunostaining for the CBPs and glutamic acid decarboxylase (GAD), it appeared that the vast majority (90%) of the Parv group was GAD, whereas only a small minority (10%) of the Calb or Calret group was GAD. Significant proportions of Calb (40%) and Calret (80%) neurons were immunopositive for phosphate-activated glutaminase, the synthetic enzyme for transmitter glutamate. The results suggested that, whereas Calret cells predominantly comprise caudally or locally projecting, possibly glutamatergic BF neurons, Parv cells likely comprise the cortically projecting GABAergic BF neurons and Calb cells the cortically projecting, possibly glutamatergic BF neurons that would collectively participate with the cholinergic cells in the modulation of cortical activity. J. Comp. Neurol. 458:11–31, 2003. © 2003 Wiley-Liss, Inc. Indexing terms: calcium binding proteins; medial septum– diagonal band; substantia innominata; preoptic nuclei

Midbrain Raphe Modulation of Nonphotic Circadian Clock Resetting and 5-HT Release in the Mammalian Suprachiasmatic Nucleus

Abstract: Serotonin (5-HT) is an important regulator of the mammalian circadian clock of the suprachiasmatic nucleus (SCN); however, critical questions remain concerning the control of serotonergic activity in the SCN and how this relates to the putative clock-resetting actions of 5-HT. Previously, we reported that electrical stimulation of the dorsal raphe nucleus (DRN) or median raphe nucleus (MRN) in hamsters evoked 5-HT release in the SCN. This DRN-stimulated 5-HT release was blocked by systemic injection of 5-HT antagonists, indicating a 5-HT receptor-mediated pathway from the DRN to the SCN. In the present study, targeted injections of the 5-HT 1,2,7 antagonist metergoline or the selective 5-HT 7 antagonist DR4004 into the DRN or MRN attenuated DRN-electrically stimulated SCN 5-HT release, supporting a multisynaptic DRN→MRN→SCN route. Intra-DRN and intra-MRN injections of the GABA A antagonist bicuculline significantly stimulated SCN 5-HT release, whereas intra-DRN or intra-MRN injections of the GABA A agonist muscimol suppressed this release. The 5-HT release induced by intra-DRN bicuculline was also blocked by co-injection of DR4004. In complementary behavioral trials, SCN 5-HT release associated with a phase-advancing sleep deprivation stimulus at midday was prevented by intra-DRN injection of metergoline. Also, phase-advance shifts induced by novel wheel access at midday were suppressed, but not blocked, by intra-DRN injection of DR4004 or muscimol. These results indicate that 5-HT 7 and GABAergic receptors of the DRN and MRN regulate behaviorally induced 5-HT release in the SCN, and that DRN output modulates nonphotic phase-resetting responses.

Coexistence of Excitatory and Inhibitory GABA Synapses in the Cerebellar Interneuron Network

Abstract: Functional GABA synapses are usually assumed to be inhibitory However, we show here that inhibitory and excitatory GABA connections coexist in the cerebellar interneuron network The reversal potential of GABAergic currents (E(GABA)) measured in interneurons is relatively depolarized and contrasts with the hyperpolarized value found in Purkinje cells (-58 and -85 mV respectively) This finding is not correlated to a specific developmental stage and is maintained in the adult animal E(GABA) in interneurons is close to the mean membrane potential (-565 mV, as measured with a novel "equal firing potential" method), and both parameters vary enough among cells so that the driving force for GABA currents can be either inward or outward Indeed, using noninvasive cell-attached recordings, we demonstrate inhibitory, excitatory, and sequential inhibitory and excitatory responses to interneuron stimulation [results obtained both in juvenile (postnatal days 12-14) and subadult (postnatal days 20-25) animals] In hyperpolarized cells, single synaptic GABA currents can trigger spikes or trains of spikes, and subthreshold stimulations enhance the responsiveness to subsequent excitatory stimulation over at least 30 msec We suggest that the coexistence of excitatory and inhibitory GABA synapses could either buffer the mean firing rate of the interneuron network or introduce different types of correlation between neighboring interneurons, or both

Olivocochlear innervation in the mouse: immunocytochemical maps, crossed versus uncrossed contributions, and transmitter colocalization.

Abstract: To further understand the roles and origins of gamma-aminobutyric acid (GABA) and calcitonin gene-related peptide (CGRP) in the efferent innervation of the cochlea, we first produced in the mouse an immunocytochemical map of the efferent terminals that contain acetylcholine (ACh), CGRP, and GABA. Olivocochlear (OC) terminals in inner and outer hair cell (IHC and OHC) regions were analyzed quantitatively along the cochlear spiral via light-microscopic observation of cochlear wholemounts immunostained with antibodies to glutamic acid decarboxylase (GAD), vesicular acetylcholine transporter (VAT), or the peptide CGRP. Further immunochemical characterization was performed in mice with chronic OC transection at the floor of the fourth ventricle to distinguish crossed from uncrossed contributions and, indirectly, the contributions of lateral versus medial components of the OC system. The results in mouse showed that (1) there are prominent GABAergic, cholinergic, and CGRPergic innervations in the OHC and IHC regions, (2) GABA and CGRP are extensively colocalized with ACh in all OC terminals in the IHC and OHC areas, (3) the longitudinal gradient of OC innervation peaks roughly at the 10-kHz region in the OHC area and is more uniform along the cochlear spiral in the IHC area, (4) in contrast to other mammalian species there is no radial gradient of OC innervation of the OHCs, and (5) all OHC efferent terminals arise from the medial OC system and terminals in the IHC area arise from the lateral OC system.

Neuropathology of prion diseases

Abstract: In prion diseases, neuropathology has remained the most important tool to give a definite diagnosis, and neuropathological research has contributed significantly to our current pathogenetic understanding. Immunohistochemistry for the disease-associated prion protein (PrP(Sc)) is indispensable for the neuropathological confirmation of prion diseases. The amount and distribution of PrP(Sc) deposits do not always correlate with type and severity of local tissue damage. PrP(Sc) deposition occurs only where neuronal parenchyma is present; in scarred infarctions with prominent gliosis, PrP(Sc) does not accumulate. Early, severe and selective loss affects a subset of inhibitory GABAergic neurons both in human and experimental prion diseases. The central pathogenetic cascade includes oxidative stress to neurons and their apoptosis. New patterns of PrP(Sc) immunoreactivity include granular ganglionic and tiny adaxonal PrP(Sc) deposits in peripheral nervous tissue, and dendritic cells and macrophages in vessel walls, suggesting that mobile haematogenous cells may be involved in spread of prions.

Anatomy of adenosine A2A receptors in brain: morphological substrates for integration of striatal function.

Abstract: A2A adenosine receptors (A(2A)Rs) are expressed with the greatest abundance in the striatum and other nuclei of the basal ganglia. The segregated expression of A(2A)Rs on the GABAergic striatopallidal medium spiny neurons, where A(2A)R and D2 dopamine receptor mRNAs are colocalized, and the opposing functional interaction between adenosine and dopamine suggest that A(2A)Rs may be an important therapeutic target. To further explore the role of A(2A)Rs in the synaptic organization of the basal ganglia, the authors developed an antibody directed against the purified A(2A)R. Immunohistochemical studies in rat brain showed dense labeling of the neuropil in the striatum, nucleus accumbens, and olfactory tubercles with lighter labeling of terminals in the globus pallidus (GP), where A(2A)R transcript is not detected. Stimulation of A(2A)Rs on GP terminals may facilitate GABAergic signaling and contribute to the overactivation observed in Parkinson's disease (PD). Analysis at the ultrastructural level allowed a more detailed characterization of the mechanism(s) of A2A-mediated control of striatal output. In the striatum, terminals expressing A(2A)Rs accounted for 25% of the labeled elements. These presynaptic receptors may facilitate excitatory glutamatergic, inhibitory GABAergic, and possibly cholinergic striatal transmission. However, the majority of striatal A(2A)R immunoreactivity was found on postsynaptic elements, including dendrites of striatopallidal neurons, in which A(2A)R and GABA immunoreactivity is colocalized. Activation of these receptors may promote GABAergic signaling in striatopallidal output neurons and their local axon collaterals in the striatum. Many of the A2A-labeled dendrites were contacted by terminals forming asymmetric (excitatory) possibly glutamatergic synapses. Using the vesicular glutamate transporters (VGLUTs) as markers of glutamatergic terminals, the authors have found that VGLUT1-immunoreactive (ir) terminals make asymmetric contacts on A2A-ir spines and spine heads in the striatum, suggesting that regulation of striatal output by A(2A)R stimulation may involve facilitation of the cortical glutamatergic excitatory input to striatopallidal neurons. These ultrastructural findings suggest several pathways through which A2A receptor blockade may act to dampen the elevated striatopallidal GABAergic signaling that occurs in PD.

Multimodal tangential migration of neocortical GABAergic neurons independent of GPI-anchored proteins

Abstract: Neuronal migration is crucial for the construction of neuronal architecture such as layers and nuclei. Most inhibitory interneurons in the neocortex derive from the basal forebrain and migrate tangentially; however, little is known about the mode of migration of these neurons in the cortex. We used glutamate decarboxylase (Gad)67-green fluorescent protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)-ergic neurons and performed time-lapse analysis. In coronal slices, many GFP-positive neurons in the lower intermediate zone (IZ) and subventricular zone (SVZ) showed robust tangential migration from lateral to medial cortex, while others showed radial and non-radial migration mostly towards the pial surface. In flat-mount preparations, GFP-positive neurons of the marginal zone (MZ) showed multidirectional tangential migration. Some of these neurons descended toward the cortical plate (CP). Intracortical migration of these neurons was largely unaffected by a treatment that cleaves glycosylphosphatidylinositol (GPI) anchors. These findings suggest that tangential migration of cortical interneurons from lateral to medial cortex predominantly occurs in the IZ/SVZ and raise the possibility that a part of the pial surface-directed neurons in the IZ/SVZ reach the MZ, whereby they spread into the whole area of the cortex. At least a part of these neurons may descend toward the CP. Our results also suggest that intracortical migration of GABAergic neurons occurs independent of GPI-anchored proteins.

Developmental Up-Regulation of KCC2 in the Absence of GABAergic and Glutamatergic Transmission

Abstract: Postsynaptic gamma-aminobutyric acid (GABA)A-mediated responses switch from depolarizing to hyperpolarizing during postnatal development of the rodent hippocampus. This is attributable to a decrease in the concentration of intracellular chloride set by the expression of the neuron-specific K+-Cl- co-transporter, KCC2. A recent in vitro study [Ganguly et al. (2001) Cell, 105, 521-532] showed that KCC2 expression may be under the trophic control of GABAA receptor-mediated transmission. Here we have studied the developmental expression of KCC2 protein in mouse hippocampal dissociated cultures as well as organotypic cultures. A low somatic expression level was found in neurons prior to the formation of the first synapses, as detected by synaptophysin immunoreactivity. Thereafter, KCC2 expression was strongly up-regulated during neuronal maturation. The developmental up-regulation of KCC2 expression was not altered by a chronic application (throughout the culturing period; 2-15 days in vitro) of the action-potential blocker TTX or the N-methyl-d-aspartate (NMDA) and non-NMDA antagonists APV and NBQX. Blockade of GABAA-mediated transmission with picrotoxin did not affect the expression levels of KCC2 protein either. These data show that neither neuronal spiking nor ionotropic glutamatergic and GABAergic transmission are required for the developmental expression of KCC2 in mouse hippocampal neurons in vitro.

Spontaneous Seizures and Loss of Axo-Axonic and Axo-Somatic Inhibition Induced by Repeated Brief Seizures in Kindled Rats

Abstract: Repeated brief seizures evoked by kindling progressively increase seizure susceptibility and eventually induce spontaneous seizures. Previous studies have demonstrated that the initial seizures evoked by kindling increase paired-pulse inhibition at 15–25 msec interpulse intervals in the dentate gyrus and also induce apoptosis, progressive neuronal loss, mossy fiber sprouting, and neurogenesis, which could potentially alter the balance of excitation and/or inhibition and modify functional properties of hippocampal circuits. In these experiments, paired-pulse inhibition in the dentate gyrus was reduced or lost after ∼90–100 evoked seizures in association with emergence of spontaneous seizures. Evoked IPSCs examined by single electrode voltage-clamp methods in granule cells from kindled rats experiencing spontaneous seizures demonstrated altered kinetics (reductions of ∼48% in 10–90% decay time, ∼40% in τ, and ∼65% in charge transfer) and confirmed that decreased inhibition contributed to the reduced paired-pulse inhibition. The loss of inhibition was accompanied by loss of subclasses of inhibitory interneurons labeled by cholecystokinin and the neuronal GABA transporter GAT-1, which project axo-somatic and axo-axonic GABAergic inhibitory terminals to granule cells and axon initial segments. Seizure-induced loss of interneurons providing axo-somatic and axo-axonic inhibition may regulate spike output to pyramidal neurons in CA3 and could play an important role in generation of spontaneous seizures. The sequence of progressive cellular alterations induced by repeated seizures, particularly loss of GABAergic interneurons providing axo-somatic and axo-axonic inhibition, may be important in the development of intractable epilepsy.

Orexin excites GABAergic neurons of the arcuate nucleus by activating the sodium--calcium exchanger.

Abstract: The neuropeptides orexins/hypocretins are essential for normal wakefulness and energy balance, and disruption of their function causes narcolepsy and obesity. Although much is known of the role of orexins in sleep/wake behavior, it remains unclear how they stimulate feeding and metabolism. One of the main targets of orexinergic neurons is the arcuate nucleus (ARC) of the hypothalamus, which plays a key role in feeding and energy homeostasis. By combining patch-clamp and RT-multiplex PCR analysis of individual neurons in mouse brain slices, we show that an electrophysiologically distinct subset of ARC neurons coexpress orexin receptors and glutamate decarboxylase-67 and are excited by orexin. Acting on postsynaptic orexin type 2 receptors, orexin activates a sodium-calcium exchange current, thereby depolarizing the cell and increasing its firing frequency. Because GABA is a potent stimulus for feeding, in both the ARC and its main projection site, these results suggest a mechanism for how orexin may control appetite.