Marc Danik

The neurobiology of apolipoproteins and their receptors in the CNS and Alzheimer's disease

The importance of apolipoproteins in the central nervous system became increasingly clear with the association in 1993 of the epsilon4 allele of apolipoprotein E with familial and sporadic late-onset Alzheimers disease. Apolipoprotein E is a ligand for several receptors, most of which are found to some extent in the brain. This review summarizes the various apolipoproteins and lipoprotein receptors found in the brain. A growing body of evidence now implicates irregular lipoprotein metabolism in several neurodegenerative disorders. We then focus on research linking apolipoprotein E and Alzheimers disease, from clinical studies to biochemical models, which may explain some of the complex neurobiology of this disorder.

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.

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.

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.

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.

Developmental and Target-Dependent Regulation of Vesicular Glutamate Transporter Expression by Dopamine Neurons

Mesencephalic dopamine (DA) neurons have been suggested to use glutamate as a cotransmitter. Here, we suggest a mechanism for this form of cotransmission by showing that a subset of DA neurons both in vitro and in vivo expresses vesicular glutamate transporter 2 (VGluT2). Expression of VGluT2 decreases with age. Moreover, when DA neurons are grown in isolation using a microculture system, there is a marked upregulation of VGluT2 expression. We provide evidence that expression of this transporter is normally repressed through a contact-dependent interaction with GABA and other DA neurons, thus providing a partial explanation for the highly restricted expression of VGluT2 in DA neurons in vivo. Our results demonstrate that the neurotransmitter phenotype of DA neurons is both developmentally and dynamically regulated. These findings may have implications for a better understanding of the fast synaptic action of DA neurons as well as basal ganglia circuitry.

Frequent coexpression of the vesicular glutamate transporter 1 and 2 genes, as well as coexpression with genes for choline acetyltransferase or glutamic acid decarboxylase in neurons of rat brain

It is widely believed that expression of the vesicular glutamate transporter genes VGLUT1 and VGLUT2 is restricted to glutamatergic neurons and that the two transporters segregate in different sets of neurons. Using single‐cell multiplex RT‐PCR (sc‐RT‐mPCR), we show that VGLUT1 and VGLUT2 mRNAs were coexpressed in most of the sampled neurons from the rat hippocampus, cortex, and cerebellum at postnatal Day (P)14 but not P60. In accordance, changes in VGLUT1 and VGLUT2 mRNA concentrations were found to occur in these and other brain areas between P14 and P60, as revealed by semiquantitative RT‐PCR and quantitated by ribonuclease protection assay. VGLUT1 and ‐2 coexpression in the hippocampal formation is supported further by in situ hybridization data showing that virtually all cells in the CA1–CA3 pyramidal and granule cell layers were highly positive for both transcripts until P14. It was revealed using sc‐RT‐mPCR that transcripts for VGLUT1 and VGLUT2 were also present in neurons of the cerebellum, striatum, and septum that expressed markers for γ‐aminobutyric acid (GABA)ergic or cholinergic phenotypes, as well as in hippocampal cells containing transcripts for the glial fibrillary acidic protein. Our study suggests that VGLUT1 and VGLUT2 proteins may often transport glutamate into vesicles within the same neuron, especially during early postnatal development, and that they are expressed widely in presumed glutamatergic, GABAergic, and cholinergic neurons, as well as in astrocytes. Furthermore, our study shows that such coexpressing neurons remain in the adult brain and identifies several areas that contain them in both young and adult rats.

Widely expressed transcripts for chemokine receptor CXCR1 in identified glutamatergic, γ‐aminobutyric acidergic, and cholinergic neurons and astrocytes of the rat brain: A single‐cell reverse transcription‐multiplex polymerase chain reaction study

Increasing evidence suggests that the chemokine interleukin (IL)‐8/CXCL8 plays important roles in CNS development, neuronal survival, modulation of excitability, and neuroimmune response. Recently, we have shown that CXCL8 can acutely modulate ion channel activity in septal neurons expressing receptors CXCR1 and/or CXCR2. This was a surprising finding, insofar as CXCR1 expression had not been described for the mammalian brain. Here we investigated whether CXCR1 transcripts are present in other brain regions, whether they are expressed at the single‐cell level in molecularly identified neurons and astrocytes, and how they are regulated during early postnatal development. In addition, possible cellular colocalization of CXCR1 and CXCR2 transcripts was examined. Semiquantitative reverse transcription‐polymerase chain reaction (RT‐PCR) revealed that CXCR1 mRNAs were expressed in the septum, striatum, hippocampus, cerebellum, and cortex (temporoparietal and entorhinal) at different levels and appeared to be regulated independently from CXCR2 during development. By using RT multiplex PCR on acutely dissociated cells from these brain regions, we show that CXCR1 transcripts were expressed in 83% of 84 sampled neurons displaying cholinergic (choline acetyltransferase mRNAs), γ‐aminobutyric acidergic (glutamic acid decarboxylases 65 and 67 mRNAs), or glutamatergic (vesicular glutamate transporters 1 and 2 mRNAs) phenotypes. CXCR1 and CXCR2 transcripts were colocalized in 45% of neurons sampled and also were present in some glial fibrillary acidic protein mRNA‐expressing astrocytes. This is the first study to demonstrate the widespread expression of CXCR1 transcripts in the brain and suggests that CXCR1 may have hitherto unsuspected roles in neuromodulation and inflammation.

Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR

Basal forebrain neurons play an important role in memory and attention. In addition to cholinergic and GABAergic neurons, glutamatergic neurons and neurons that can corelease acetylcholine and glutamate have recently been described in the basal forebrain. Although it is well known that nerve growth factor (NGF) promotes synaptic function of cholinergic basal forebrain neurons, how NGF affects the newly identified basal forebrain neurons remains undetermined. Here, we examined the effects of NGF on synaptic transmission of medial septum and diagonal band of Broca (MS-DBB) neurons expressing different neurotransmitter phenotypes. We used MS-DBB neurons from 10- to 13-d-old rats, cultured on astrocytic microislands to promote the development of autaptic connections. Evoked and spontaneous postsynaptic currents were recorded, and neurotransmitters released were characterized pharmacologically. We found that chronic exposure to NGF significantly increased acetylcholine and glutamate release from cholinergic MS-DBB neurons, whereas glutamate and GABA transmission from noncholinergic MS-DBB neurons were not affected by NGF. Interestingly, the NGF-induced increase in neurotransmission was mediated by p75NTR. These results demonstrate a previously unidentified role of NGF and its receptor p75NTR; their interactions are crucial for cholinergic and glutamatergic transmission in the septohippocampal pathway.