| Background Itch,also known as pruritus,is a common symptom caused by a variety of skin or systemic diseases,usually resulting in an unpleasant sensation and accompanied with scratching.So far,the underlying mechanism of the itch signal transmission remains unclear.The recent researches mainly focus on the peripheral nervous system to the diencephalon of the central nervous system.However,whether itch information is transmitted from the thalamus to the cerebral cortex is still not fully understood.Among the whole brain regions activated by acute itch stimulation,the paraventricular thalamic nucleus(PVT)is one of the most activated brain regions.PVT is an important nucleus in the midline nucleus of the thalamus.Morphological evidence suggests that PVT sends projections to the limbic system,including the medial prefrontal cortex(m PFC),insular cortex(IC),cingulate cortex(CC),nucleus accumbens(NAc),etc,suggesting that the PVT may be involved in complex functional activities through these fiber connections.Collateral projection is commonly found in the central nervous system and could transmit information to two or more areas at the same time.They are one of the mechanisms of neural information integration.Although it has been reported that PVT sends collateral projections to the central amygdaloid nucleus(Ce A),NAc and bed nucleus of the stria terminalis(BNST),whether the same mechanism exists between PVT and the limbic system remains unclarified.m PFC,IC and Ce A are the three nuclei that form the densely connecting with PVT in the limbic system(LS).Results of functional magnetic resonance imaging(f MRI)indicate increased blood oxygen level dependent(BOLD)level of m PFC,IC and Ce A under acute itch stimulation.However,whether the neurons that transmit itch information from PVT to m PFC,IC and Ce A are collateral projection neurons has not been demonstrated.Objective To probe whether the PVT sent collateral projections to the m PFC and IC,m PFC and Ce A,IC and Ce A in the mouse.Furthermore,acute itch model induced by histamine was used in mice to examine whether these collateral projections participate in itch transmission.This study provides morphological basis for thalamic-cortical connection involved in itch sensation information transmission,and provides new ideas for elucidating itch information transmission and regulation mechanism in higher centers.Methods In the present study,morphological methods were utilized to examine the possible connections of PVT and limbic system,and their modulatory roles in itch sensation.Firstly,m PFC,IC and Ce A were chosen as target nuclei through effluent projection screening results.The acute itch model was then performed to testify the involvement of these PVT collateral projection neurons in itch signal transmission.The contents of the present work are divided into four parts: 1.Neural fiber connections between PVT and m PFC,IC Ce A were observed using tracing methods The r AVV2/9-h Syn-e GFP and r AVV2/R-h Syn-m Cherry were injected into PVT to examine the neural connectivity of PVT and limbic system.The retrograde tracer fluoro-gold(FG)was injected into the m PFC,IC and Ce A of mice,respectively,and the distribution of FG-labeled neurons in PVT were observed by immunohistochemical staining.2.PVT innervated the m PFC/IC,m PFC/Ce A,IC/Ce A by way of their axonal collaterals Mice were perfused 7 d after FG and cholera toxin subunit B(CTB)injections into the m PFC and IC,m PFC and Ce A,IC and Ce A,respectively.Immunofluorescence histochemical staining was applied to examine the distributions of the collateral projection neurons,which were mainly observed in ipsilateral PVT.3.The projection from the PVT to m PFC,IC,Ce A of the mouse expressed FOS with the help of fluorescence immunostaining Mice were intradermally injected with 15 μl histamine to induce an acute itch model.Significant more FOS-immunoreactive(FOS-ir)neurons were observed in PVT than that in the control group,suggesting PVT activation in acute itch model.4.The collateral projection from the PVT to m PFC/IC,m PFC/Ce A,IC/Ce A of the mouse expressed FOS by using fluorescence immunostaining FG/CTB/FOS triple-labeled staining was applied,which demonstrated that collateral projection neurons in the PVT sent itch signals to m PFC/IC,m PFC/Ce A and IC/Ce A,respectively.Results 1.Bidirectional neural fiber connections between PVT and m PFC,IC,Ce A were observed by neural tract tracing r AVV2/9-h Syn-e GFP was injected into the PVT.A lot of e GFP-labeled fibers could be observed in the prefrontal cortex(PFC),nucleus accumbens(NAc),cingulate cortex(CC),claustrum(CLA),and Ce A.Meanwhile,after r AVV2/R-h Syn-m Cherry was injected into PVT,many m Cherry-labled projection neurons in the PFC,CC,IC and Ce A can be observed.The number of FG-labeled neurons in PVT were 143.32 ± 33.21,133.33 ± 10.00 and 131.67 ± 40.57,respectively,after FG was injected into the right m PFC,IC,and Ce A of mice.The proportion of m PFC,IC,Ce A projection neurons were(52.25 ± 5.58)%,(39.97 ± 6.30)%,(62.00 ± 10.90)% in rostral PVT,and(47.75 ± 5.58)%,(60.03 ± 6.30)%,(38.00 ± 10.90)% in caudal PVT,suggesting that the number of m PFC,IC and Ce A projection neurons in PVT were similar,but they were different in the distribution of the rostral-caudal PVT.2.PVT innervated the m PFC/IC,m PFC/Ce A,IC/Ce A by way of their axonal collaterals CTB and FG were injected into different nuclei(m PFC/IC,m PFC/Ce A and IC/Ce A)on the right side of mice,respectively,and the number of collateral projections neurons from PVT to m PFC/IC,m PFC/Ce A and IC/Ce A were 18.36 ± 2.00,10.11 ± 5.21 and 47.82 ± 12.25,respectively.Meanwhile,the proportion of collateral projections neurons were(15.41 ± 5.30)%,(4.42 ± 1.01)% and(18.42 ± 3.81)% in rostral PVT,(8.84 ± 0.58)%,(5.77 ± 1.07)% and(14.56 ± 1.95)% in caudal PVT,respectively.3.Involvement of the projection from the PVT to m PFC,IC,Ce A of the mouse expressed FOS In His-induced acute itch model,the number and proportion of m PFC,IC and Ce A projection neurons activated in PVT were 19.32 ± 11.25,46.21 ± 25.78,39.00 ± 1.63,and(12.16 ± 4.89)%,(33.26 ± 9.59)%,(32.16 ± 8.43)%,respectively,suggesting that efferent projections from the PVT to IC or Ce A might be closely interacted with itch information.4.Involvement of the collateral projection from the PVT to m PFC/IC,m PFC/Ce A,IC/Ce A of the mouse expressed FOS The number and proportion of neurons that project from PVT to m PFC/IC,m PFC/Ce A and IC/Ce A were 2.31 ± 1.24,3.10 ± 2.54,8.36 ± 2.00 and(11.94 ± 3.24)%,(23.33 ± 3.33)%,(17.50 ± 7.50)%,respectively.In conclusion,acute itch stimulation caused a higher proportion of FOS protein expressed to m PFC/Ce A projection neurons,suggesting that they play an important role in the transmission of itch information.Conclusion 1.The paraventricular thalamic nucleus(PVT)transmits itch information to the m PFC,IC and Ce A of the limbic system by bidirectional fiber connections.2.PVT innervates the m PFC/IC,m PFC/Ce A and IC/Ce A by way of their axonal collaterals,and the projection neurons to the m PFC/Ce A express a higher proportion of FOS,suggesting that they are crucial in the transmission of itch information.3.The number of projection neurons to the IC/Ce A in the dorsolateral part of the PVT is the largest in PVT,indicating that the neurons in the dorsolateral part of the PVT mainly transmit itch information to different regions of the limbic system by their axonal collaterals. |
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