Chemical phenotypes of MS/DBB neurons identified by sc-RT-PCR
A total of 106 Medial Septal/Diagonal Band Complex (MS/DBB) neurons were electrophysiologically recorded and their chemical phenotypes were identified by multiplex sc-RT-PCR. The results showed that the mRNAs encoding nestin, ChAT, glutamic acid decarboxylases 67 (GAD67
), vesicular glutamate transporters 1 or 2 (VGLUT1
) could be reversely transcribed and amplified from the harvested cytoplasm. Automatic sequencing confirmed that each PCR product is from the target cDNA. The MS/DBB neurons studied in our experiment were comprised of 79 ChAT mRNA-positive neurons (ChAT+) that are cholinergic neurons, 13 GAD67
mRNA-positive neurons (GAD67
+) that are GABAergic neurons. There were 11 neurons co-expressing ChAT mRNA and GAD67
mRNA, of which, 6 were categorized as cholinergic neuron and 5 as GABAergic neuron according to their electrophysiological properties. Nine neurons solely expressed VGLUT1
mRNA or/and VGLUT2
mRNAs but not ChAT mRNA or GAD67
mRNAs, which confirms the identification of glutamatergic neurons [4
]. Among the 40 nestin mRNA-positive (nestin+) neurons, 87.5% (35/40) neurons expressed ChAT mRNA. Conversely, 44.3% (35/79) of ChAT+ neurons expressed nestin mRNA. However, no nestin mRNA was found co-expressing GAD67
mRNA or VGLUT mRNA. The neurons did not express any of these mRNA were discarded.
The intrinsic membrane properties of nestin mRNA+ neurons
Eighty-seven neurons were assessed for their electrophysiological profile. This assessment identified 69 slow-firing neurons, 5 cluster-firing neurons and 13 fast-firing neurons. Although some neurons presented rebound action potentials following a hyperpolarization current injection, we did not find any typical burst-firing neuron in our experiment. All of the recorded nestin+ cells were excitable, with typical electrophysiological characteristics of functional neurons. In voltage-clamp mode, when depolarized from -60 mV, typical neuronal whole-cell currents (comprised of large inward Na+ current and outward K+ current) could be elicited (Figure ). In current-clamp mode, typical neural action potential was observed in response to a short period of depolarization, and repetitive action potentials could be elicited when sustained positive current was applied (Figure ). As most of nestin mRNA co-expressed with ChAT mRNA, we first compared electrophysiological properties of nestin+ neurons (including nestin mRNA-positive & ChAT mRNA-negative neurons and nestin mRNA-positive & ChAT mRNA-positive neurons) to nestin mRNA-negative & ChAT mRNA-positive (nestin- & ChAT+) neurons, and with GAD67+ neurons and VGLUT+ neurons (Table , Figures , and ). Then, we compared electrophysiological properties of nestin mRNA-positive & ChAT mRNA-negative (nestin+ & ChAT-) neurons, nestin mRNA-positive and ChAT mRNA-positive (nestin+ & ChAT+) neurons, and nestin mRNA-negative & ChAT mRNA-positive neurons (nestin- & ChAT+) so as to identify the electrophysiological characteristics among different categories of nestin and ChAT expressing patterns (Figures and ).
Figure 1 Nestin mRNA-positive cells in MS/DBB are functional neurons. A. Agarose gel analysis of the sc-RT-PCR products obtained from a single MS/DBB cell. The only PCR-generated fragment was nestin. B. Whole cell current of the same cell depolarized from -60 (more ...)
Electrophysiological properties of chemically identified neurons in the rat medial septal/diagonal band complex
Figure 2 Comparison of action potential properties of MS/DBB neurons. A. Representative action potentials from all cell types (superimposed) show the differences in spike shape and width among the four cell types. GAD67+ neurons have the narrowest action potentials, (more ...)
Figure 3 Electrophysiological properties of nestin+ and/or ChAT+ neurons. A1-F1: Nestin+ & ChAT- neurons, A2-F2: Nestin+ & ChAT+ neurons, A3-F3: Nestin- & ChAT+ neurons. A1-3: Agarose gel analysis of the sc-RT-PCR products to identify chemical (more ...)
Figure 4 Electrophysiological properties of GAD67+ and VGLUT+ neurons. A1-F1: GAD67+ neuron presents electrophysiological properties of fast-firing neuron. A2-F2: VGLUT+ neuron presents electrophysiological properties of cluster-firing neuron. A1-2: Agarose gel (more ...)
Comparison of RMP and Ih amplitude of nestin+ and/or ChAT+ neurons in MS/DBB. * P < 0.05; ** P < 0.01.
Nestin+ neurons (including those neurons co-expressing ChAT mRNA) had a mean fire rate (MF) of 8.39 ± 0.45 Hz, maximal firing frequency (FMAX) of 20.73 ± 2.07 Hz, and steady firing frequency (FSTEADY) of 6.86 ± 0.53 Hz. These neurons also had broad action potentials (spike width 2.46 ± 0.09 ms) and large afterhyperpolarization (duration 223.77 ± 12.08 ms, amplitude 4.49 ± 0.21 mV), which were similar to nestin- & ChAT+ neurons (P > 0.05). These data suggest that nestin+ neurons share many basic characteristics with nestin- & ChAT+ neurons in the MS/DBB. The spike width, AHP amplitude and duration of nestin+ neurons were significantly larger than that of GAD67+ neurons (P < 0.01). However, MF, depolarizing sag, and hyperpolarization activated current of neurons (Ih) were smaller than those of GAD67+ neurons (P < 0.01). Furthermore, other key properties of nestin+ neurons were significantly different from those of GAD67+ neurons (P < 0.05, Table , Figure ). Thus, the nestin+ neurons were distinctive from the classic GABAergic neurons. Nestin+ neurons shared some membrane properties of VGLUT+ neurons, but had larger Ih and no cluster firing in response to prolonged depolarization from -60 mV. In summary, the Ih of nestin+ neurons were smaller than those of the GAD67+ neurons, but greater than the Ih of the VGLUT+ (Figures and ). Statistical analysis showed the different Ih and other parameters among the subpopulations of neurons in MS/DBB (Table1).
Interestingly, while further analyzed the electrophysiological properties of nestin+ & ChAT-, nestin+ & ChAT+ and nestin+ & ChAT- neurons, we found that the Ih of nestin+ & ChAT+ neurons were larger than those of nestin- & ChAT+ neurons (P < 0.05), and nestin+ & ChAT- neurons had a RMP of -51.80 ± 1.32 mV, which were significantly lower than that of nestin- & ChAT+ neurons (P < 0.01) (Figures and ). However, other electrophysiological differences (e.g., latency for first spike, slow after-hyperpolarizing potential, maximal frequency and action potential decay slope) among these neurons were not found.
Excitatory postsynaptic currents recorded from nestin+ & ChAT+ neurons and nestin- & ChAT+ neurons
In this section, nineteen neurons, which contained 12 nestin- & ChAT+ neurons and 7 nestin+ & ChAT+ neurons, were recorded, and 3993 sEPSCs events and 3570 mEPSCs events were analyzed in total. The addition of 10 μM 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, a non-NMDA glutamate receptor antagonist) abolished all synaptic events, indicating the involvement of non-NMDA glutamate receptors (data not shown).
The sEPSCs amplitude (28.45 ± 1.78 pA) of nestin+ & ChAT+ neurons was significantly larger than that of the nestin- & ChAT+ neurons (22.91 ± 1.05 pA) (student's t-
test, two tails, P
< 0.05). The sEPSCs amplitudes cumulative probability distribution curve of nestin+ & ChAT+ neurons showed a right shift compared to the curve for nestin- & ChAT+ neurons (K-S Z = 4.549, P
< 0.01). This result suggests that the sEPSCs distribution patterns of nestin+ & ChAT+ neurons were different from those of nestin- & ChAT+ neurons. It also provided further evidence to confirm that the sEPSCs amplitude of nestin+ & ChAT+ neurons was significantly larger than those of the nestin- & ChAT+ neurons. Both the student's t
-test and the Kolmogorov-Smirnov test (KS-test) were used to determine if the two datasets differ significantly. As student's t
-test is a parametric test and may be more sensitive if the data meets the requirements of the student's t
-test. The KS-test, on the other hand, has the advantage of making no assumptions about the distribution of data (non-parametric). Therefore, in order to compare the mean value and distribution of the sEPSCs and mEPSCs, we used both, finding that the KS-test is more suited than the student's t
-test. The sEPSCs inter-event intervals cumulative probability distribution curve of nestin+ & ChAT+ neurons was on the left of the curve for nestin- & ChAT+ neurons (K-S Z = 2.644, P
< 0.01), which indicates the sEPSCs frequency of the nestin+ & ChAT+ neurons was higher than that of the nestin- & ChAT+ neurons (Figure ) [23
Figure 6 Comparison of sEPSCs of MS/DBB nestin- and nestin+ cholinergic neurons. A. Consecutive traces of sEPSCs recorded from MS/DBB nestin- and nestin+ cholinergic neurons. B. Average sEPSCs of nestin- and nestin+ cholinergic neurons. C. Comparison of the sEPSCs (more ...)
In order to further explore the mechanism of the different sEPSCs between the nestin- & ChAT+ and nestin+ & ChAT+ neurons, mEPSCs of both kinds of neurons were studied. The independent samples student's t-test showed that the mEPSCs amplitude (29.01 ± 1.83 pA) of nestin+ & ChAT+ neurons was significantly larger than that of nestin- & ChAT+ neurons (22.64 ± 1.06 pA) (P < 0.01). The mEPSCs amplitudes cumulative probability distribution curve of nestin+ & ChAT+ neurons was on the right of that for the nestin- & ChAT+ neurons (K-S Z = 8.2165, P < 0.01). The mEPSCs inter-event intervals cumulative probability distribution curve of nestin+ & ChAT+ neurons was on the right for that of nestin- & ChAT+ neurons (K-S Z = 1.717, P < 0.01). These results confirmed that nestin+ & ChAT+ neurons had higher mEPSCs amplitude than nestin- & ChAT+ neurons and that the mEPSCs frequency of nestin+ & ChAT+ neurons was lower than that of the nestin- & ChAT+ neurons (Figure ).
Figure 7 Comparison of mEPSCs of MS/DBB nestin- and nestin+ cholinergic neurons. A. Consecutive traces recorded from MS/DBB nestin- and nestin+ cholinergic neurons. B. Average mEPSCs of nestin- and nestin+ cholinergic neurons. C. Comparison of the mEPSCs frequencies (more ...)
The paired samples student's t-test results showed that the mEPSCs frequency was significantly lower than sEPSCs frequency in nestin+ & ChAT+ (P < 0.05), but no difference was found between the mEPSCs and sEPSCs frequencies of nestin- & ChAT+ neurons (P > 0.05). The sEPSCs/mEPSCs frequency ratio of nestin+ & ChAT+ neurons was approximately two times higher than that nestin- & ChAT+ neurons. However, there was no difference between the amplitudes of mEPSCs and sEPSCs of nestin- & ChAT+ neurons or nestin+ & ChAT+ neurons (P > 0.05). These results suggest that the higher sEPSCs amplitude of nestin+ & ChAT+ neurons compared to the nestin- & ChAT+ neurons was not changed by 1 μM TTX, implied it might come from the higher excitability of nestin+ & ChAT+ neurons themselves rather than from stronger excitatory action potentials of presynaptic neurons. Furthermore, there was no difference between synaptic multiplicities of nestin+ & ChAT+ neurons and nestin- & ChAT+ neurons, which suggests that the nestin+ & ChAT+ neurons and nestin- & ChAT+ neurons shared similar maturity (Figure ). In summary, these results provide powerful evidence that despite shared similar maturity, nestin+ & ChAT+ neurons receive stronger excitatory synaptic inputs and have higher excitability compared to nestin- & ChAT+ neurons.
Figure 8 Comparison of sEPSCs and mEPSCs of nestin- and nestin+ cholinergic neurons in MS/DBB. A. Comparison of the frequencies of sEPSCs and mEPSCs of nestin- and nestin+ cholinergic neurons. B. Comparison of the amplitude of sEPSCs and mEPSCs of nestin- and (more ...)
Immunofluorescence study of the biocytin-filled neurons
Twenty-eight neurons were successfully filled with biocytin and visualized by Rhodamine Red-X. Cell bodies were particularly well-labelled, allowing us to determine their position relative to the MS/DBB. Biocytin-filled neurons were bipolar or multipolar and gave off two or three primary dendrites that subsequently bifurcated to the adjacent areas. The axons originated from the soma or proximal end of a primary dendrite. No evidence of axon collaterals was found in our slices. Of the 28 biocytin-filled neurons, 22 were ChAT-immunoreactive (ChAT-ir) neurons, among which 45.45% (10/22) were nestin-immunoreactive (nestin-ir) neurons. Eleven out of the 28 biocytin-filled neurons were nestin-ir neurons, 90.91% (10/11) of which were also ChAT-immunoreactive (Figure ).
Figure 9 Triple immunofluorescent study of biocytin-filled neuron. A. Biocytin-filled neuron was visualized by rhodamine red-X-conjugated streptavidin. B and C showed ChAT- and nestin-immunoreactive neurons visualized by cy2 (blue) and alexa 405 (green) respectively. (more ...)
Retrograde tracing of fast blue from the CA1 area of hippocampus
Examination of serial section of the basal forebrain region 5 days after injection of the fast blue revealed that the blue colour fluorescence could be visualized clearly. The fast blue labelled somas were seen throughout the entire MS/DBB region. Histological examination of the MS/DBB area after labelling revealed striking intense signals in the cell body, however, the neuritis were difficult to distinguish from background. In order to define the anatomical circuits of the nestin+ and nestin- cholinergic projection to the hippocampus, we evaluated the percentage of ChAT and nestin immunoreactivity among the fast blue-labelled neurons in the MS/DBB region after fast blue intra-hippocampus instillation. The nestin and ChAT immunoreactive cells were clearly labelled by green and red colour fluorescence specifically. In order to find the ratio of the nestin+ or nestin- cholinergic neurons projection to the hippocampus, the double or triple fluorescence of combined immunohistochemistry and retrograde labelling were carefully measured. Approximately 20.40% of the fast blue-labelled neurons in the MS/DBB area were ChAT-immunoreactive. In which, 48.04% were nestin-expressing neurons, and 51.96% nestin non-expressing cholinergic neurons (Figure ).
Figure 10 Retrograde labelling demonstrates that the nestin+ and nestin- cholinergic neurons projected to hippocampus. (A) Photomicrograph demonstrating the deposition of fast blue dye throughout the entire MS/DBB area and the location of ChAT+ and nestin+ neurons. (more ...)