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.

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.

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 GABA A 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.