Figure 6.6. T h e power spectra of the wave terms of two example cells which were putatively identified as GABAergic (top) and cholinergic (bottom). T h e power o f a cell's wave form was measured at 16 ordinates between OHz and the critical sampling frequency o f 25kHz. Calibration bars: 1msec, lOOpV.
this would show up on the power spectrum as a secondary peak distinct from the lower frequency main peak corresponding to the basic spike. As figure 6.6 shows, cells with a hump in their repolarisation phase instead produced a distinct broadening of the primary peak. The difference between the two spectral patterns was sufficiently large, however, to allow the cells to be split into two groups. This was done by examining the proportional difference between the values of the second and third ordinates. Those in which the third ordinate was more than 3/4 of the second were assigned to the putative s-
AHP/cholinergic group, and the rest to the putative f-AHP/GABAergic group.
Figure 6.7 demonstrates that this method yielded a fairly clear separation between the two types of cells. As some cells were recorded over more than one day, and the grouping of records from different days was performed by comparison of action potential wave forms, this graph uses the ordinate ratio of each day's record to avoid inadvertent corruption of the data. 34 cells (53 records) were assigned to the cholinergic group, and 36 cells (59 records) to the GABAergic group. The average number of daily records per cell is very similar between the two groups (1.56 versus 1.64 respectively), suggesting that this did not introduce any notable bias. In only two cases did records assigned to the same cell fall into different categories. In these cases, the classification of the cell was decided by visual inspection of the wave form.
As figure 6.8 shows, this classification did produce two groups with a
significantly different firing behaviour distribution (%^=14.3, d.f=4, p<0.01). One third (12/36) of the putative GABAergic cells belonged to type lb (firing rhythmic, phase-locked bursts of one or a few spikes), whereas 44% (15/34) of
Page 194 0 0 1 5 “O c o u <v to T 3 C 03 to O) 3 fC > c O) O l g O l _Q 03 putative GABAergic cells putative cholinergic cells
Figure 6.7. A plot of the ratio between the values o f the third and second ordinates o f the wave form power spectrum. As the hand-fitted curves show, it is plausible that these measurements reflect two separate underlying distributions, which allow the cells to be divided into two groups at a criterion ratio of 0.75.
□ CABAn
lb ic
Cell Type
Figure 6.8. GABA and cholinergic cells, sorted by examination of their action potential wave form spectrum, manifested significant differences in terms of their firing behaviour (%2=I4.3, d.f.=4, p<0.01). The cell type refers to the classification o f Gaztelu and Buno (1982), as detailed in the previous chapter and describes the cells' relationship with hippocampal theta rhythm. Type 1 cells fire rhythmic bursts, type 2 cells are significantly phase-locked to theta rhythm, but fail to show rhythmicity in their autocorrelogram, and type 3 cells are non-rhythmic.
"ACh"
5 9 13 17 21 25 2 9 33 37 41 4 5 4 9 53 5 7 61
"GABA"
■ Jl iL L
5 9 13 17 21 25 29 33 37 41 4 5 4 9 53 5 7 61
Figure 6.9. T he distributions of the mean firing rates o f daily cell records. The cells were assigned to the cholinergic or GABAergic groups by examination of the spectrum o f their action potential wave forms.
Page 196
4 -
3 ^
2
1
Putative cholinergic cells
10 3 0 50 70 90 n o 1 3 0 150 170 190 2 1 0 2 3 0 2 5 0 2 7 0 2 9 0 3 1 0 3 3 0 3 5 0
phase (degrees)
Putative GABAergic cells
10 3 0 50 70 9 0 1 1 0 130 150 170 190 2 1 0 2 3 0 2 5 0 2 7 0 2 9 0 3 1 0 3 3 0 3 5 0
phase (degrees)
Figure 6.10. T he preferential phases o f M S/DBB cells after they had been classified as cholinergic or GABAergic by examination o f the wave form spectrum. There is no evidence o f any systematic distribution.
the putative cholinergic cells belonged to type 2 (displaying a significant theta phase lock without rhythmic bursting or noticeable rate modulation). There was no difference, however, in the distribution of average firing rates between the two types of cells (figure 6.9). Neither did either of the two cell groups display any overall preferential phase (figure 6.10).
D iscu ssio n
It is important to note that the reliability of the classification presented here is not entirely certain. In separating the cells on the basis of a “hump” in the repolarisation phase of the action potential, this method is indirectly
determining the presence of a slow AHP in the recorded cell. This, in and of itself, does not, of course, guarantee the type of neurotransmitter used by the cell’s projections, although the current evidence (as discussed previously) does suggest that this criterion can be used to make broad distinctions. It should be noted as well that Matthews and Lee themselves found a degree of uncertainty in classifying cells on the basis of their intracellular observations.While many of the cells that were found could easily be assigned to one of the two groups, 42% of the fast-AHP cells had action potential lengths which overlapped with those of the slow-AHP cells, and some fast-AHP cells failed to fire any faster than the slow-AHP cells.
Despite these caveats, however, the data presented here provide good evidence for a fundamental difference in the firing behaviours of GABAergic and
cholinergic cells, namely that GABAergic cells tend to fire rhythmic bursts (type lb), whereas cholinergic cells tend to non-rhythmic firing which still shows some significant (albeit lesser) theta phase modulation (type 2). As
__________________________________________________________________________ Page 198
demonstrated previously (chapter four), there is a significantly higher number of type 2 cells in awake, freely moving animals compared to the anaesthetised condition. This suggests that one of the differences between MS/DBB cell function in the anaesthetised and awake states is a higher degree of phasic modulation of cholinergic cells in the latter case. At present, it is still not possible to say whether this modulation arises from brainstem inputs, from changes in rhythmic interactions within the MS/DBB, or from the inhibitory backprojeétions from the hippocampus.
These data contrast with the conclusions reached by Stewart and Fox (1989a), who discovered that systemic injection of atropine caused one group of
rhythmically bursting MS/DBB cells to fire in an incoherent, non-rhythmical manner, while a second group was unaffected. The authors assumed that the atropine-sensitive cells were cholinergic and the atropine-resistant ones GABAergic, although there is anatomical evidence that GABAergic cells receive cholinergic synapses (Léranth and Frotscher, 1989), and that nearly all GABAergic and cholinergic cells carry muscarinic cholinergic receptors (van der Zee and Luiten, 1994). As the data presented here show that the majority of rhythmically bursting (type la and lb) cells are probably GABAergic, it is more likely that the atropine-sensitive cells recorded by Stewart and Fox were a subgroup of the GABAergic population.
Another important implication of the low rhythmicity of cholinergic cells is that it contradicts the model of Stewart and Fox (1990) in which cholinergic septo-hippocampal projections cause fast, rhythmic excitation of hippocampal interneurons. The non-rhythmic, type 2 firing behaviour of MS/DBB
cholinergic effects (Benardo and Prince, 1982) makes it unlikely that cholinergic projections are important in relaying rhythmic impulses. Although direct
intracellular recordings of an interneuron’s response to cholinergics have not been performed, it has been shown that application of carbachol causes an increase in the number of spontaneous IPSPs recorded in pyramidal cells in a hippocampal slice preparation. This result implies that cholinergics do indeed excite hippocampal interneurons. This excitation has an onset latency of several seconds, however, which is far too slow for rhythmic transmission at the 8-9Hz theta rhythm frequency found in rats. It must be concluded, therefore, that cholinergic MS/DBB cells do not directly pace hippocampal cells to produce theta rhythm, but that their effect is exerted over a longer timeframe.