Figure 1.
Muti-tetrode recording and VTA neuron classification.
(A) Electrode array track shown on an example coronal brain section (top-right) and locations of the electrode array tips (from 21 mice) on atlas section diagrams [52]. Blue squares represent the locations where type-1/2 putative DA neurons were recorded; red squares represent the locations where type-3 neurons were recorded; purple squares represent the locations where both type-1/2 and type-3 neurons were recorded (see Figure 2 for the classification of the three types of putative DA neurons). (B) Examples of typically recorded spike waveforms for putative DA (red) and Non-DA (blue and black) neurons. Half AP width was measured from the trough to the following peak of the action potential. (C) Baseline firing rates and half AP widths of the classified DA (red) and Non-DA (black) neurons. DA, dopamine; Non-DA, non-dopamine; AP, action potential.
Figure 2.
Three types of VTA putative dopamine (DA) neurons.
(A-C) Peri-event rasters (1-20 trials, from top to bottom) and histograms of three examples of VTA putative dopamine neurons (A: type-1, B: type-2, and C: type-3) in response to free fall (left panels), shake (middle panels), and the conditioned tone that reliably predicted sugar pellet delivery (right panels). (D) Percentages of different types of putative DA neurons. (E, F) Percentages of fear-suppressed (E: type-1 and 2) and fear-excited (F: type-3) putative DA neurons that were significantly activated by the conditioned tone that reliably predicted sugar pellet delivery. Free fall, 30 cm high; Shake, 0.2 sec; Tone, 5 kHz, 1 sec.
Figure 3.
Firing patterns and pharmacology characterizations.
(A–C) Three examples of tetrode-recorded putative dopamine neurons (type-1, type-2, and type-3) and their representative spike waveforms. PC1 and PC2 represent the first and second principal components in the principal component analysis, respectively. Blue dots represent individual spikes for the isolated dopamine neurons; black dots indicate individual spikes for other unsorted VTA neurons. (D) Inter-spike intervals of three examples of putative dopamine neurons (type-1, type-2, and type-3). (E) Percentages of burst firing for the three types of putative dopamine neurons. Error bars, s.e.m.; ***P<0.001, Student's t-test. (F) Baseline firing rates of the three types of putative dopamine neurons. Error bars, s.e.m.; ***P<0.001, Student's t-test. (G) Cumulative spike activity of thee examples of putative dopamine neurons (type-1, type-2, and type-3) in response to the dopamine receptor agonist apomorphine. It was noted that the type-1 and type-3 putative dopamine neurons were recorded simultaneously from one tetrode. (H and I) Baseline and post-drug firing rates of putative dopamine (H) and non-dopamine (I) neurons. Mice were injected with the dopamine receptor agonist apomorphine (1 mg/kg, i.p.) and the firing rates were averaged 30 min before and 30 min after apomorphine injection.
Figure 4.
Responses of VTA type-1 putative dopamine neurons to different durations and intensities of fearful events.
(A) Peri-event rasters (1–20 trials) and histograms of one example type-1 neuron in response to 10 cm (left) and 30 cm (right) free fall events. (B) Smoothed population average peri-event histograms (left) and offset excitation latencies (right) of type-1 neurons in response to 10 cm (blue line; n = 15) and 30 cm (red line; n = 20) free fall events. (C) Peri-event rasters and histograms of another type-1 neuron in response to 0.5 sec (left) and 1 sec (right) shake events. (D) Smoothed population average peri-event histograms (left) and offset excitation latencies (right) of type-1 neurons in response to 0.2 sec (green line; n = 13), 0.5 sec (blue line; n = 20), and 1 sec (red line; n = 14) shake events. (E) Peri-event rasters and histograms of another type-1 neuron in response to low-(left) and high-intensity (right) shake events. (F) Smoothed population average peri-event histograms (left) and offset excitation peak firing rates (right) of type-1 neurons in response to low- (blue line; n = 9) and high-intensity (red line; n = 9) shake events. Error bars, s.d.; *P<0.05, ***P<10-8, Student's t-test.
Figure 5.
Responses of VTA type-3 dopaminegic-like neurons to different durations and intensities of fearful events.
(A) Peri-event rasters (1-20 trials) and histograms of one example type-3 neuron in response to 10 cm (left) and 30 cm (right) free fall events. (B) Smoothed population average peri-event histograms (left) and offset excitation latencies (right) of type-3 neurons in response to 10 cm (blue line; n = 8) and 30 cm (red line; n = 10) free fall events. (C) Peri-event rasters and histograms of the same neuron (as shown in A) in response to 0.5 sec (left) and 1 sec (right) shake events. (D) Smoothed population average peri-event histograms (left) and offset excitation latencies (right) of type-3 neurons in response to 0.2 sec (green line; n = 10), 0.5 sec (blue line; n = 9), and 1 sec (red line; n = 7) shake events. (E) Peri-event rasters and histograms of another type-3 neuron in response to low- (left) and high-intensity (right) shake events. (F) Smoothed population average peri-event histograms (left) and offset excitation peak firing rates (right) of type-3 neurons in response to low- (blue line; n = 5) and high-intensity (red line; n = 5) shake events. Error bars, s.d.; *P<0.05, ***P<10-5, Student's t-test.
Figure 6.
Bi-directional encoding of positive and negative signals via the same conditioned tone in different contexts.
(A) Schematic of experimental paradigm for bidirectional conditioning. One tone (5 kHz, 1 sec) was used throughout: it predicted sugar pellet delivery in the reward chamber (top); it predicted free fall event in the free-fall chamber (middle); and it did not predict anything in the neutral chamber (bottom). (B) Left, delay of dish approach after onset of the conditioned tone that predicted sugar delivery. Right, mice showed significant increased backward movement after onset of the conditioned tone that predicted free fall event. (C) Aversion-like behaviors (frequent defecation and urination) elicited in the free-fall chamber compared with the reward or neutral chamber. Error bars, s.e.m.; n = 10; *P<0.05, **P<0.01, ***P<0.001, Student's t-test. (D, E) Peri-event rasters (1–20 trials) and histograms of two examples of VTA putative dopamine neurons in response to the same conditioned tone that predicted sugar pellet delivery (left), that predicted free fall event (middle), and that did not predict anything (right), with an interval of 1–2 h between sessions. (F) Smoothed population average peri-event histograms of fear-suppressed (type-1 and 2) putative dopamine neurons in response to the same conditioned tone that predicted sugar pellet (left panel; n = 16), that predicted free fall event (middle panel; the same 16 neurons as shown in the left panel), and that did not predict anything (right panel; n = 10). Free fall, 30 cm high.
Figure 7.
Responses of type-3 dopaminergic-like neurons to positive and negative signals via the same conditioned tone in different contexts.
(A) Peri-event rasters (1–20 trials) and histograms of an example type-3 neuron in response to the same conditioned tone that predicted sugar pellet delivery (left), that predicted free fall event (middle), and that did not predict anything in the neutral chamber (right). (B) Smoothed population average peri-event histograms of type-3 neurons (n = 6) in response to the same conditioned tone that predicted sugar pellet delivery (left), that predicted free fall event (middle), and that did not predict anything (right). Free fall, 30 cm high.
Figure 8.
Response onset latencies of the VTA putative dopamine neurons.
(A) Response onset latencies of individual type-1 and 2 dopamine neurons to free fall and shake events. (B) Response onset latencies of individual type-3 dopamine neurons to free fall and shake events. (C) Response onset latencies of individual type-1 and 2 dopamine neurons to the reward CS that predicted sugar pellet and the aversive CS that predicted free fall. (D) Response onset latencies of individual type-3 dopamine neurons to the reward CS that predicted sugar pellet and the aversive CS that predicted free fall. (E) Population average response onset latencies of type-1 and 2 dopamine neurons (from the same data as shown in A and C) and (F) type-3 neurons (from the same data as shown in B and D). Response onset latencies for type-1/2 neurons to free fall, shake and aversive CS correspond to the latencies of suppression; while the others correspond to the latencies of activation. Error bars, s.d.
Figure 9.
Synchrony among unique sets of VTA putative dopamine neurons.
(A) Peri-event rasters (1–20 trials) and histograms of two simultaneously recorded type-1 neurons in response to free fall event, and (B) the cross-correlogram between these two neurons when the mouse was freely behaving. (C) The averaged cross-correlograms between simultaneously recorded type-1 neurons (48 pairs during freely behaving, and 35 pairs during sleep). (D) Peri-event rasters and histograms of two simultaneously recorded type-1 and type-2 neurons in response to free fall event, and (E) the cross-correlogram between these two neurons during freely behaving. (F) The averaged cross-correlograms between simultaneously recorded type-1 and type-2 neurons (6 pairs during both freely behaving and sleep). (G) Peri-event rasters and histograms of two simultaneously recorded type-3 neurons in response to free fall event, and (H) the cross-correlogram between these two neurons during freely behaving. (I) The averaged cross-correlograms between simultaneously recorded type-3 neurons (15 pairs during freely behaving, and 12 pairs during sleep). (J) Peri-event rasters and histograms of two type-1 and type-3 neurons (simultaneously recorded from one tetrode) in response to free fall event, and (K) the cross-correlogram between these two neurons during freely behaving. (L) The averaged cross-correlograms between simultaneously recorded type-1 and type-3 neurons (12 pairs during freely behaving, and 10 pairs during sleep). Free fall, 30 cm high.