Sylvain Williams

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

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.

Self-generated theta oscillations in the hippocampus

Hippocampal theta rhythm is crucial for spatial memory and is thought to be generated by extrinsic inputs. In contrast, using a complete rat hippocampus in vitro, we found several intrinsic, atropine-resistant theta generators in CA1. These oscillators were organized along the septotemporal axis and arose independently from CA3. Our results suggest that CA1 theta rhythm can emerge from the coupling of multiple autonomous hippocampal theta oscillators.

Dopamine neurons in culture express VGLUT2 explaining their capacity to release glutamate at synapses in addition to dopamine

Dopamine neurons have been suggested to use glutamate as a cotransmitter. To identify the basis of such a phenotype, we have examined the expression of the three recently identified vesicular glutamate transporters (VGLUT1‐3) in postnatal rat dopamine neurons in culture. We found that the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3. In comparison, serotonin neurons express only VGLUT3. Single‐cell RT‐PCR experiments confirmed the presence of VGLUT2 mRNA in dopamine neurons. Arguing for phenotypic heterogeneity among axon terminals, we find that only a proportion of terminals established by dopamine neurons are VGLUT2‐positive. Taken together, our results provide a basis for the ability of dopamine neurons to release glutamate as a cotransmitter. A detailed analysis of the conditions under which DA neurons gain or loose a glutamatergic phenotype may provide novel insight into pathophysiological processes that underlie diseases such as schizophrenia, Parkinsons disease and drug dependence.

The chemokine interleukin-8 acutely reduces Ca2+ currents in identified cholinergic septal neurons expressing CXCR1 and CXCR2 receptor mRNAs

The chemokine IL‐8 is known to be synthesized by glial cells in the brain. It has traditionally been shown to have an important role in neuroinflammation but recent evidence indicates that it may also be involved in rapid signaling in neurons. We investigated how IL‐8 participates in rapid neuronal signaling by using a combination of whole‐cell recording and single‐cell RT‐PCR on dissociated rat septal neurons. We show that IL‐8 can acutely reduce Ca2+ currents in septal neurons, an effect that was concentration‐dependent, involved the closure of L‐ and N‐type Ca2+ channels, and the activation of Giα1 and/or Giα2 subtype(s) of G‐proteins. Analysis of the mRNAs from the recorded neurons revealed that the latter were all cholinergic in nature. Moreover, we found that all cholinergic neurons that responded to IL‐8, expressed mRNAs for either one or both IL‐8 receptors CXCR1 and CXCR2. This is the first report of a chemokine that modulates ion channels in neurons via G‐proteins, and the first demonstration that mRNAs for CXCR1 are expressed in the brain. Our results suggest that IL‐8 release by glial cells in vivo may activate CXCR1 and CXCR2 receptors on cholinergic septal neurons and acutely modulate their excitability by closing calcium channels.

Causal evidence for the role of REM sleep theta rhythm in contextual memory consolidation

Let sleeping mice remember The role of REM (rapid eye movement) sleep for memory consolidation has been discussed for a long time. Boyce et al. used optogenetics to inhibit theta oscillations in the mouse hippocampus during REM sleep (see the Perspective by Kocsis). Both object recognition memory and contextual fear memory were impaired. This consolidation mechanism occurred in a critical time window immediately after training. Disrupting the same system for similar durations during non-REM sleep or wakefulness had no effect on memory. Science, this issue p. 812; see also p. 770 Selectively disrupting REM sleep impairs hippocampus-dependent memory formation. Rapid eye movement sleep (REMS) has been linked with spatial and emotional memory consolidation. However, establishing direct causality between neural activity during REMS and memory consolidation has proven difficult because of the transient nature of REMS and significant caveats associated with REMS deprivation techniques. In mice, we optogenetically silenced medial septum γ-aminobutyric acid–releasing (MSGABA) neurons, allowing for temporally precise attenuation of the memory-associated theta rhythm during REMS without disturbing sleeping behavior. REMS-specific optogenetic silencing of MSGABA neurons selectively during a REMS critical window after learning erased subsequent novel object place recognition and impaired fear-conditioned contextual memory. Silencing MSGABA neurons for similar durations outside REMS episodes had no effect on memory. These results demonstrate that MSGABA neuronal activity specifically during REMS is required for normal memory consolidation.

A functional glutamatergic neurone network in the medial septum and diagonal band area

The medial septum and diagonal band complex (MS/DB) is important for learning and memory and is known to contain cholinergic and GABAergic neurones. Glutamatergic neurones have also been recently described in this area but their function remains unknown. Here we show that local glutamatergic neurones can be activated using 4‐aminopyridine (4‐AP) and the GABAA receptor antagonist bicuculline in regular MS/DB slices, or mini‐MS/DB slices. The spontaneous glutamatergic responses were mediated by AMPA receptors and, to a lesser extend, NMDA receptors, and were characterized by large, sometimes repetitive activity that elicited bursts of action potentials postsynaptically. Similar repetitive AMPA receptor‐mediated bursts were generated by glutamatergic neurone activation within the MS/DB in disinhibited organotypic MS/DB slices, suggesting that the glutamatergic responses did not originate from extrinsic glutamatergic synapses. It is interesting that glutamatergic neurones were part of a synchronously active network as large repetitive AMPA receptor‐mediated bursts were generated concomitantly with extracellular field potentials in intact half‐septum preparations in vitro. Glutamatergic neurones appeared important to MS/DB activation as strong glutamatergic responses were present in electrophysiologically identified putative cholinergic, GABAergic and glutamatergic neurones. In agreement with this, we found immunohistochemical evidence that vesicular glutamate‐2 (VGLUT2)‐positive puncta were in proximity to choline acetyltransferase (ChAT)‐, glutamic acid decarboxylase 67 (GAD67)‐ and VGLUT2‐positive neurones. Finally, MS/DB glutamatergic neurones could be activated under more physiological conditions as a cholinergic agonist was found to elicit rhythmic AMPA receptor‐mediated EPSPs at a theta relevant frequency of 6–10 Hz. We propose that glutamatergic neurones within the MS/DB can excite cholinergic and GABAergic neurones, and that they are part of a connected excitatory network, which upon appropriate activation, may contribute to rhythm generation.

Glutamatergic Neurons of the Mouse Medial Septum and Diagonal Band of Broca Synaptically Drive Hippocampal Pyramidal Cells: Relevance for Hippocampal Theta Rhythm

Neurons of the medial septum and diagonal band of Broca (MS-DBB) provide an important input to the hippocampus and are critically involved in learning and memory. Although cholinergic and GABAergic MS-DBB neurons are known to modulate hippocampal activity, the role of recently described glutamatergic MS-DBB neurons is unknown. Here, we examined the electrophysiological properties of glutamatergic MS-DBB neurons and tested whether they provide a functional synaptic input to the hippocampus. To visualize the glutamatergic neurons, we used MS-DBB slices from transgenic mice in which the green fluorescent protein is expressed specifically by vesicular glutamate transporter 2-positive neurons and characterized their properties using whole-cell patch-clamp technique. For assessing the function of the glutamatergic projection, we used an in vitro septohippocampal preparation, electrically stimulated the fornix or chemically activated the MS-DBB using NMDA microinfusions and recorded postsynaptic responses in CA3 pyramidal cells. We found that glutamatergic MS-DBB neurons as a population display a highly heterogeneous set of firing patterns including fast-, cluster-, burst-, and slow-firing. Remarkably, a significant proportion exhibited fast-firing properties, prominent Ih, and rhythmic spontaneous firing at theta frequencies similar to those found in GABAergic MS-DBB neurons. Activation of the MS-DBB led to fast, AMPA receptor-mediated glutamatergic responses in CA3 pyramidal cells. These results describe for the first time the electrophysiological signatures of glutamatergic MS-DBB neurons, their rhythmic firing properties, and their capacity to drive hippocampal principal neurons. Our findings suggest that the glutamatergic septohippocampal pathway may play an important role in hippocampal theta oscillations and relevant cognitive functions.

Behavioral characterization of dysbindin-1 deficient sandy mice

Dysbindin-1 (dystrobrevin binding protein-1) has been reported as a candidate gene associated with schizophrenia. Dysbindin-1 mRNA and protein levels are significantly reduced in the prefrontal cortex and hippocampus of schizophrenia subjects. To understand the in-vivo functions of dysbindin-1, we studied schizophrenia relevant behaviors in adult male Sandy homozygous (sdy/sdy) and heterozygous (sdy/+) mice that have a natural mutation in dysbindin-1 gene (on a DBA/2J background) resulting in loss of protein expression. Spontaneous locomotor activity of sdy/sdy and sdy/+ mice in novel environment was not significantly different from DBA/2J controls. However, on repeated testing in the same environment for 7 days, sdy/sdy mice, in contrast to DBA/2J controls showed a lack of locomotor habituation. Locomotor activating effect of a low dose of d-amphetamine (2.5 mg/kg i.p.), a behavioral measure of mesolimbic dopamine activity, was significantly reduced in the mutant mice. Interestingly, sdy/sdy mice showed enhanced locomotor sensitization to repeated five daily injection of amphetamine. Possible cognitive impairment in Sandy mutants was revealed in novel object recognition test as sdy/sdy and sdy/+ mice spent significantly less time exploring novel objects compared to DBA/2J. Sdy/sdy mice also showed deficits in emotionally motivated learning and memory showing greater freezing response to auditory conditioned stimulus (CS) in fear conditioning paradigm. In thermal nociceptive test, the latency of paw withdrawal in sdy/sdy and sdy/+ animals was significantly higher compared to DBA/2J indicating hypoalgesia in the mutants. Taken together, these data suggest that dysbindin-1 gene deficiency leads to significant changes in cognition and altered responses to psychostimulants.

Phosphorylation of tau protein at sites Ser396–404 is one of the earliest events in Alzheimer's disease and Down syndrome

Phosphorylation, conformational changes and cleavage of tau protein have been widely suggested to contribute to abnormal tau processing in the pathogenesis of Alzheimers disease, as well as in other tauopathies. Consistently, many phosphorylated sites, such as Ser199–202–Thr205 and Ser396–404, have been associated with this pathological processing. The present study examined the chronological appearance of phosphorylation during the neurofibrillary tangle (NFT) evolution in Alzheimer disease (AD) and Down syndrome.

The Hippocamposeptal Pathway Generates Rhythmic Firing of GABAergic Neurons in the Medial Septum and Diagonal Bands: An Investigation Using a Complete Septohippocampal Preparation In Vitro

The medial septum diagonal band area (MS/DB) projects to the hippocampus through the fornix/fimbria pathway and is implicated in generating hippocampal theta oscillations. The hippocampus also projects back to the MS/DB, but very little is known functionally about this input. Here, we investigated the physiological role of hippocamposeptal feedback to the MS/DB in a complete in vitro septohippocampal preparation containing the intact interconnecting fornix/fimbria pathway. We demonstrated that carbachol-induced rhythmic theta-like hippocampal oscillations recorded extracellularly were synchronized with powerful rhythmic IPSPs in whole-cell recorded MS/DB neurons. Interestingly, we found that these IPSPs evoked rebound spiking in GABAergic MS/DB neurons. In contrast, putative cholinergic and glutamatergic MS/DB neurons responded only weakly with rebound spiking and, as a result, were mostly silent during theta-like oscillations. We next determined the mechanism underlying the rebound spiking that followed the IPSPs in MS/DB GABAergic neurons using phasic electrical stimulation of the fornix/fimbria pathway. We demonstrate that the increased rebound spiking was attributable to the activation of Ih current, because it was significantly reduced by low concentrations of the Ih antagonist ZD7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride]. Together, these results suggest that rhythmical activity in hippocampus is transferred to the MS/DB and can preferentially phase the spiking of GABAergic MS/DB neurons because of their significant expression of Ih currents. Our data demonstrate that hippocamposeptal inhibition facilitates theta rhythmic discharges in MS/DB GABAergic neurons while favoring the inhibition of most ACh and glutamate neurons.