By histochemistry for cytochrome oxidase, nuclear demarcations around the POm can be clearly identified. Therefore, after penetration tract was identified by HRP-DAB method, as shown in Fig 6, cell locations can be identified under this histology processes.
Schematic diagrams shown in Fig 20 illustrate cell locations. Each symbol represents different response categories to mechanical stimulation. The diagrams show that the majority of responsiveness units were located in the POm-VP border, which is consistent to our tracing results.
Discussion
The most important findings of the present study are: (1) The massive incerto-POm projection may tonically inhibit POm tactile and nociceptive functions.
(2) The hidden nociceptive POm function was revealed in ZI lesioned condition.
From our tracing studies, we found that incerto-thalamic fibers project densely to the POm in the border region to the VP, in which is convergent with peripheral nociceptive inputs. And from our electrophysiological results, POm responses to innocuous and noxious stimulation are revealed by ZI lesion. These data demonstrate that the ZI neurons may modulate the processing of nociceptive information in the POm.
Incerto-thalamic terminal field
Our tracing results showed that the ZI projects densely to the POm. Despite POm, thalamic nuclei containing terminals from ZI included ipsilateral side of centrolateral nucleus (CL), ventrolateral thalamic nucleus (VL), angular thalamic nucleus (AngT), laterodorsal thalamic nucleus (LD), reticular thalamic nucleus (Rt) and ZI itself. None of the first order thalamic nucleus of the dorsal thalamus was labeled in this study.
The results are consistent to previous findings (Power et al., 1999; Bartho et al., 2002;
Cavdar et al., 2006).
In all of the tracing cases in present study, focused and limited ZI injections
resulted in abundant fibers labeling within the ZI itself, which indicates the property of intranuclear connections among ZI neurons. Comparing to the Rt, another ventral thalamic nucleus giving inhibitory control to the POm, the ZI seems to have more intranuclear connections (Bartho et al., 2002). Previous data is also mentioned that there is a network of interconnections between the ZI and the higher- order thalamic nuclei on both sides of the thalamus (Power and Mitrofanis, 1999, 2001). In our studies, we also examined the labeled profiles of contralateral side. However, no labeled terminal was discovered on the contralateral side. Smaller amount of tracer deposits in the present study might be a reason for the inconsistency.
Comparing with previous data about projection distribution regions from the superficial lamina of dorsal horn (Iwata et al., 1992; Gauriau and Bernard, 2004a), we found that there is a convergent area in the POm around the VP-POm border from the peripheral nociceptive input and the ZI input. These results indicate that the region is highly involved in nociception processing in anatomical grounds. Therefore, we determined the VP-POm border area to be the main target in our electrophysiological study.
Nociceptive function of POm
From our electrophysiology study, we found that there are more POm units in the ZI lesion side responded to innocuous and noxious stimulation comparing with the
contralateral side or naïve animals, showing that some POm units may convey noxious mechanical or noxious heat information after releasing form the ZI inhibition.
And the most responsive area is concentrated on the VP-POm boarder, as demonstrated in our anatomical tracing results.
Considering the coding ability for stimulation intensity, we found that POm units may be as good as VP units in mechanical stimulation after ZI lesion. It is now widely accepted that pain is composed of two components: sensory-discriminative and affective-motivational components (Melzack and Casey, 1968). According to the theory, discriminative information, such as stimulus intensity and location, is transmitted through the dorsal horn to the VP and eventually to the somatosensory cortex. Medial thalamus is thought to be involved in affective or motivational aspects of pain due to irregular responses to noxious stimuli and large receptive fields (Bushnell and Duncan, 1989). Therefore, in the present study, we try to test the hypothesis that POm units may release from the ZI inhibition by examination the coding ability for mechanical and thermal stimulation. We found that responses of POm units also show a linear responses to increasing stimuli intensity comparing to the VP, which is believed involving in discriminative component of pain processing.
Anatomical evidence shows that lemniscal and spinothalamic system, which both consist in the discriminative system, terminate in the POm (Jones, 2007). Therefore,
we concluded that the POm might be involved in discriminative aspects of pain.
In addition to the POm recording, VP responses were also recorded in this study for positive control and for comparison purpose. From our anatomical results and previous findings, it is clear that incerto-thalamic fibers selectively terminate in the POm but VP (Power et al., 1999; Bartho et al., 2002). In the present study, we found no significant difference between tactile responsive units in the VP of the ZI lesion side versus those of the intact side, which is consistent to this connectional data.
However, there is obviously a lack of numbers in VP, which is not consistent with previous findings (Chiang et al., 2005). This may be due to the bias of the recording tracts in the present study. According to the previous anatomical findings, superficial lamina of dorsal horn would terminate more laterally and ventrally in the VP (Iwata et al., 1992; Gauriau and Bernard, 2004a). However, our penetration tracts were more medially and dorsally in order to record the POm. Therefore, bias penetration tracts might be a consequence for encountering less noxious-responsive units in VP.
Although ZI projects densely to the POm, some POm units still didn’t respond to peripheral stimuli after ZI lesion. Unresponsive units in the POm after ZI lesion may represent another population of units that receive other nuclei modulation instead of ZI. Indeed, the Rt is also reported to project to the POm (Pinault and Deschenes, 1998;
Pinault, 2004). Previous reports have even demonstrated a new GABAergic pathway from anterior pretectal nucleus (APT) to the POm with ultrastructural features similar to those of ZIv afferents (Bokor et al., 2005).
Hypotheses for ZI modulation
The present study proposes that the relay of noxious inputs in the higher-order thalamic nuclei, such as POm, relies on a mechanism of disinhibition from the ZI.
That is, inhibition of the inhibitory incerto-thalamic pathway. This proposal raises an issue: under what conditions is ZIv active or inactive, thus inhibiting or releasing the flow of signals through POm. There are mainly two hypotheses proposed recently.
State-dependent gating hypothesis indicates that inhibition of ZIv is mediated through brainstem cholinergic neurons that increase their firing frequency at arousal state (Trageser et al., 2006). According to this hypothesis, increasing cholinergic activity during wakefulness would suppress ZI-mediated inhibition, thereby permitting POm responses to peripheral stimuli. In support of this hypothesis, stimulation of brainstem cholinergic neurons in anesthetized animals was found to suppress excitability of ZI neurons and to enhance sensory transmission in POm (Masri et al., 2006; Trageser et al., 2006).
Another hypothesis proposed is that sensory transmission in POm is mediated by a top-down disinhibitory mechanism which is contingent on motor activity. In support
of this hypothesis, stimulation of the motor cortex was found to suppress the vibrissal responses in ZIv. An intra-incertal GABAergic circuitry was found to mediate the suppression (Urbain and Deschenes, 2007).
Both of the hypotheses described above provide proposals for POm disinhibition in intact animals. In other words, POm may convey nociceptive information in intact, awake, and free-moving animals when disinhibited from the tonic ZI control.
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Tables and Figures
Table 1: Response categories to mechanical stimulation of the POm units.
Significant differences were between the ZI-lesion side to the contralateral side, and ZI-lesion side to the POm of naïve animals (p <0.01, chi-square test).
Unit number of POm Response
Category ZI-lesion side Contralateral side Naïve rat
Tactile+ Pinch+
6
(10 %)0 0
Tactile- Pinch+
10
(17 %)0 1
(2 %)Tactile+ Pinch-
13
(22 %)3
(9 %)6
(10 %)Tactile- Pinch-
30
(51 %)29
(91 %)52
(88%)Total
59 32 59
Table 2: Response categories to mechanical stimulation of the VP units.
No significant difference among groups (Chi-square test).
Unit number of VP Response
Category ZI-lesion side Contralateral side Naïve rat
Tactile+ Pinch+
1
(3 %)0 3
(5 %)Tactile- Pinch+
2
(7 %)0 8
(14 %)Tactile+ Pinch-
12
(44 %)11
(78 %)19
(35 %)Tactile- Pinch-
12
(44 %)3
(21 %)24
(44 %)Total
27 14 54
Table 3: Response categories to contact heat stimulation of the POm units.
Significant differences were between the ZI-lesion side to the contralateral side, and ZI-lesion side to the POm of naïve animals (p <0.05, chi-square test).
Unit number of POm Response
Category ZI-lesion side Contralateral side Naïve rat Heat +
14
(45 %)3
(16 %)10
(22 %)Heat
-17
(55 %)16
(84 %)35
(78 %)Total
31 19 45
Table 4: Converge and segregation of thermal nociceptive and mechanical nociceptive inputs in POm.
Mechanical and thermal stimulations were both tested in the units of this table.
Unit number of POm Response
Category ZI-lesion side Contralateral side Naïve rat
Heat+ Pinch+
3
(9 %)0 1
(2 %)Heat+ Pinch-
7
(22 %)3
(16 %)3
(8 %)Heat- Pinch+
0 0 0
Heat- Pinch-
21
(67 %)15
(83 %)34
(89 %)Total
31 18 38
Table 5: Response categories to contact heat stimulation of the VP units.
No significant difference among groups (Chi-square test).
Unit number of VP Response
Category ZI-lesion side Contralateral side Naïve rat
Heat +
1
(4 %)1
(7 %)2
(7 %)Heat
-23
(96 %)13
(93 %)28
(93 %)Total
24 14 30
Figure 1: A 16-channel Michigan probe used in the electrophysiology experiments. A:
Appearance of the electrode. B: An enlarged view of the tip of the electrode. Distance between each recording site is 50 μm. Scale bar in A: 5 mm, and in B: 100μm.
A B
Figure 2: An example of a ZI neuron showing wide-dynamic range responsiveness to mechanical stimulation. A: Nissl-stained coronal section showing an electrolytic lesion (asterisk) at the recording site in ZI. B: Cumulative waveforms of the neuron.
C: A 3D view of cluster analysis with PC1-PC2-PC3 plot of spikes of this neuron (black cluster). D: The receptive field (gray area) of this neuron. E: The responses of this neuron to mechanical stimuli (A: airpuff, B: brush, T: tapping, and P: pinch).
Scale bar in A: 1 mm, and in B: 100 μs. Bin size in E: 1 s.
B A
*
C
D E
Time (s)
Frequency (ips)
A B T P
0 5 10 15 20
0 200 400 600
Figure 3: An example of responsiveness units along a ZI recording tract at the level of P 4 mm and L 2.5 mm referring to the bregma. A: The anatomical relationship around the ZI at the level of electrode penetration site. B: Depths of the locations of units encountered and their respective receptive fields. The dashed line separates receptive fields observed in the VP (above the dashed line) and the ZI (below the dashed line).
ic, internal capsule; ml, medial lemniscus; Rt, reticular thalamic nucleus; STh, subthalamic nucleus; VP, ventral posterior nucleus; ZI, Zona incerta; No RFs: no prominent receptive field was observed. Scale bar: 0.5 cm.
A B
5.4 nose/whiskers
6.2 no RFs 6.4 whiskers
6.8 forepaw VP
ml
STh ic ZI
Rt
Figure 4: Coronal section stained with cresyl violet (B) and another example
immunohistochemistry for parvalbumin (C, D) one week after kainic acid lesions of the ZI unilaterally. A complete loss of neurons and gliosis was discernible at the lesion side (B, D). A: The camera lucida drawing illustrates the anatomical relationship around the ZI and the extent of the lesions (gray area). C: The contralateral side of the ZI can be distinguished to the dorsal (ZId) and the ventral portion (ZIv) by
immunohistochemistry for parvalbumin. Scale bar in A and B: 500 μm; in C and D:
250 μm. ZId: dorsal sector of Zona incerta; ZIv: ventral sector of Zona incerta. See other abbreviations in Figure 3 legend.
D
ic STh
B
VP
ml ZI
ic
A
STh
ZId
C
STh ic
ZIv
Figure 5: Diagram of stimulation protocol. A: Mechanical stimulation. B: Contact thermal stimulation. Gray bars indicating the stimulus duration is 15 s in A, and 30 s in B.
B
35 ℃ 40 ℃ 35 ℃ 43 ℃ 35 ℃ 45 ℃ 35 ℃ 50 ℃ 35 ℃
3 min 30s 3 min 3 min 3 min 3 min
A
30 s 15 s 30 s 15 s
Brush Pinch Brush Pinch
5 min
Figure 6: A: An example of the Michigan probe penetration tract (arrow) in a cytochrome oxidase (CO) stained coronal section. Arrow indicates the electrolytic lesion at the deepest recording site. B: Response category of each unit of the channel which is represented in A. The dashed line separates the densely CO immunoreactive VP (below the dashed line) and the lightly CO immuoreactive POm (above the dashed line). Scale bar in A: 200 um. POm, posterior medial thalamic nucleus; VP, ventral posterior nucleus; NR, unit not responsive to any of the stimuli tested. T+P-: Tactile positive, pinch negative unit.
A
10 9 8Figure 7: A: One example of BDA injected in the ZI whiskers area. Photomicrograph of a coronal section shows the injection site. B: Photomicrograph of BDA-labeled terminals in the ipsilateral POm. The dashed line separates the POm and the VP.
Amplified terminals boutons are shown in the insert. C: Coronal planes showing the distribution of BDA-labeled terminals in the ipsilateral thalamus. Scale bar in A: 200 μm, in B: 100 μm ,in B insert: 2μm, and in C: 0.5 cm. See figure 3 legend for other abbreviations.
C A
- 2.5 mm - 3 mm - 3.3 mm
- 3.5 mm - 3.8 mm - 4.3 mm
VP
B
POmFigure 8: Summary of the labeled terminals distributions after BDA injected into the ZI whiskers (n = 4) and forepaw (n = 1) regions. A: BDA injection sites in the ZI. B: Color-coded terminal fields. Each color represents results from one rat. The numerical numbers in figure B are
anteroposterior stereotaxic coordinates of the respective coronal sections relative to bregma. Scale bar in A: 200 μm, and in B: 0.5 cm. See figure 3 ic
VP ml
STh
A B
- 2.5 mm
POm
VL VP LD
CL
- 3 mm - 3.3 mm
- 4.3 mm - 3.8 mm
- 3.5 mm
POm VP
Figure 9: Input convergence area in the POm from the superficial laminae of dorsal horn and ZI. Data from the dorsal horn is modified from nucleus caudalis (Iwata et al., 1992) and superficial lamiae of dorsal horn of lumbosacral enlargement (Gauriau and Bernard, 2004a).
From Zona incerta
From superficial laminae of dorsal horn
- 3.3 mm - 3.5 mm - 3.8 mm
Figure 10: Camera lucida drawings of the ZI lesion sites of the seven rats recorded.
Gray areas indicate the extent of the leision. ic, interal capsule; VP, ventral posterior nucleus; ZI, Zona incerta. Scale bar: 0.5 cm.
VP
ZI ic
Figure 11: An example of a POm neuron showing low-threshold responsiveness to mechanical stimulations. A: Cumulative waveforms of the neuron. B: The location of the unit. C: The receptive field (gray area) of this neuron. D: The responses of this neuron to mechanical stimuli. Scale bar in A: 100 μs. Bin size in D: 1 s.
A B C
D
0 5 10 15
0 30 60 90 420 450 480 510
Brush Pinch
Time (s)
Frequency (ips)
Brush Pinch POm
VP
Figure 12: An example of a POm neuron showing wide-dynamic range
responsiveness to mechanical stimulations. A: Cumulative waveforms of the neuron.
B: The location of the unit. C: The receptive field (gray area) of this neuron. D: The responses of this neuron to mechanical stimuli. Scale bar in A: 100 μs. Bin size in D:
1 s.
A B C
D
Brush Pinch Brush Pinch
Time (s)
Frequency (ips)
420 450 480 510
0 10 20 30 40
0 30 60 90 120
POm
VP
Figure 13: An example of a POm neuron of the naïve animal responses to two trials of mechanical stimulation. The neuron shows high-threshold responsiveness (arrow) to the first trial of mechanical stimulation, while it showed no responsiveness to the second trial. It is therefore defined as a “Tactile- Pinch-” neuron. Bin size: 1 s.
Brush Pinch
Time (s)
Frequency (ips)
420 450 480 510
0 2 4 6 8
0 30 60 90 120
Brush Pinch
Figure 14: Response properties of the VP units with or without the ZI lesion. Left panel represents a unit recorded in the VP with the ZI lesion ipsilaterally, middle panel represents one recorded in the contralateral side, and right panel represents the other one recorded in the VP without the ZI lesion. The units locations are shown
Figure 14: Response properties of the VP units with or without the ZI lesion. Left panel represents a unit recorded in the VP with the ZI lesion ipsilaterally, middle panel represents one recorded in the contralateral side, and right panel represents the other one recorded in the VP without the ZI lesion. The units locations are shown