| BACKGROUND&OBJECTIVEIn creatures,fear is one of the most important and essential emotion characterize for survival and evolution,in order to maintain their survival and reproduction,the danger from the outside world can inspire creatures for defense.However,fear like panic was not necessary for the creatures.Pavlovian fear conditioning is the archetypal paradigm used to study the acquisition,storage,retrieve and retention of fearful memories.In a laboratory setting,fear is acquired when an innocuous stimulus(the conditioned stimulus(CS),i.e.an auditory cue)is paired with a noxious stimulus(the unconditioned stimulus(US),i.e.foot shock)such that following several trials,the CS presentation alone produces the learned fear response(the conditioned response(CR),e.g.,freezing).Over subsequent unpaired presentations of the CS,the fear abates in a process termed extinction.But for the fear conditioning training animals,CR showed significant reduction with unpaired presentations of the CS.Without fear extinction training,a significant increase in CR expression with the extension of the time interval of CR,the fear can resurface after a period of recovery time,it means the original fear memory remains intact.This fear abates does not mean that eliminate or forget the established CS-US links,but the formation of a new learning and memory CS-no US.The fear abates may process the newly created CS-no US gradual memory enhancement,extinction entails learning a new "safe" memory that competes with or inhibits the fear memory.The original fear memory remains intact,and this means the PTSD(post-traumatic stress disorder,PTSD)patients will break out repeatedly.The PTSD patients who experienced a major catastrophic event or cause serious mental trauma,the patients have long been plagued by persistent mental disorders.Traumatic experiences repeatedly reproduce,escape the traumatic event and increased alertness.This will be a terrorist incident,such traumatic memories will reproduce in the mind day after day,affected the normal life and work seriously,its essence is a condition associated with the traumatic event fear memory reproduction.Such as PTSD soldiers returning from the battlefield may be link the roar of helicopters to particular severe traumatic experiences,the sound of helicopters will cause intense fear reaction.Currently PTSD patients primarily take exposure therapy,exposure refers to the patients face their fears to stimulate not followed the aversive stimulus,patients get used to fear,so the gradual elimination of the fear response,unfortunately,due to the original fear memory remains intact,the fear encountered when CS can be restored.The fear can resurface when the CS is encountered in an environment outside of the extinction context.This limits the efficacy of exposure therapy.Fear extinction memory acquisition,consolidation and extraction of brain regions involved in memory and fear of the same brain regions,but may involve different neural circuits.Neurobiological basis of conditioned fear response is believed to be an important reason for the fear subsided difficult.Therefore,in hopes of identifying targets for intervention,many investigations have focused on revealing the neural substrates of fear extinction.Behavioral investigations of extinction have identified three main neural regions:the basolateral amygdala(BLA),the medial prefrontal cortex[(MPF:comprised of the prelimbic(PL),infralimbic(ILA),anterior cingulate(ACA)cortical areas],and the hippocampus(HPF).The amygdala is attached at the end of the hippocampus,which is a part of the limbic system.It plays an important role in emotional expression,especially in the fear expression and locates anterior temporal lobe,lateral lower corners of the top edge,deep surface parahippocampal gyrus,connects to the end of the caudate nucleus.BLA is a part of the amygdala and is a major internal sensory information interface,which receives and integrates information input from different parts of the sensory information,including from the geniculate body,the primary auditory cortex,the entorhinal cortex,insular cortex and the hippocampus(including CA1,piriform cortex,subiculum and entorhinal cortex).All the information in the BLA was integrated and transmit to the fear response region CEA,and then projected into different areas of the brain stem and hypothalamus to produce different reactions,for example,it projected to PAG produce freezing reaction,projected to the pons reticular nucleus produce startle reflex,projected to the lateral hypothalamus,the dorsal vagal nucleus will makes heartbeat faster,high blood pressure and other cardiovascular reactions,projected to the parabrachial nucleus so breathing will slower and other respiratory reactions,projecting to hypothalamic paraventricular nucleus and bed nucleus stria terminalis so that the hormone level will increase.BLA is not only involved in the expression of fear,but also the fear extinction.More and more behavioral evidence identified that BLA involved in fear extinction memory acquisition,consolidation and recall.Converging behavior evidence implicates the ILA is involved in extinction recall and the PL in extinction expression.So the network with MPF's nervous leading to fear of expression or extinction gain more attention,a series of studies implicated that connections between MPF and HPF are involved in these functions.MPF receives excitatory projections from the HPF,and HPF is the most important brain region involved in the regulation of contextual memory.How all of these structures and their subdivisions are interconnected in a network with other brain regions to regulate the behavior remains relatively unknown.Interpretations of these functional data without a well-defined structural substrate are limited due to the fact that important subdivisions during manipulations of these structures for behavior studies may be overlooked.Therefore,reconstructing the neural network for extinction will help to guide and constrain testable hypotheses regarding the roles of these structures and their subdivisionsin the behavior.METHODSThe connections of a particular region are studied in relation to other brain regions in order to establish how a multitude of structures work in concert to govern behavior.This can be accomplished by identifying all afferents,efferents,and reciprocal connections of a given brain region.Intermediate areas through which a structure communicates with other brain regionsare also important.The double coinjection method used simultaneously reveals all of this information.It entails injecting two anatomically delineated brain regions with a unique mixture of one anterograde and one retrograde tracer in a single animal.Double coinjections of phaseolus vulgaris leucoagglutinin(anterograde)with cholera toxin b subunit(retrograde)(PHAL/CTb)and biotinylated dextran amine(anterograde)with Fluoro gold(retrograde)(BDA/FG)were used.The double coinjection method entails making two separate injections into anatomically delineated areas in a single animal.Each injection is a mixture of one anterograde(PHAL or BDA)and one retrograde tracer(CTb or FG).Anterograde tracers like PHAL(green)and BDA(red)are picked up by the soma and transported to the terminal boutons thereby revealing the efferents of the injection site,while retrograde tracers like CTb(magenta)and FG(gold)are taken up by the terminals and transported back to the somata revealing the afferents of the injection site.Reciprocity between the injection site and a target region is indicated by the overlap of PHAL and CTb or by BDA and FG.Intermediate stations between the two injection sites are exposed by the overlap of PHAL and FG or BDA and CTb.To chart the fear extinction network,data from injections of neuronal tracers made into different regions of the cortex,amygdala,hippocampal formation,and caudoputamen were analyzed.By coinjections the anterograde tracer and retrograde tracer has been connected with the anatomical pathways of fear extinction,but whether these connections are functional.By means of cytogenetics,we can confirm these functional circuits more precisely.RESULTS1.The mBLAa connections to the prefrontal regionOur data show thatthe BLAa has strong bidirectional connections with both the ILA and PL and that inputs from the amygdala to the ILA and PL originate selectively from the BLAa and from no other amygdalar nucleus.When a coinjection(BDA/FG)is made into the PL,anterogradely labeled BDA fibers and retrogradely labeled FG cells overlap in the medial part of the BLAa(mBLAa)showing a strong reciprocal mBLAa(?)PL connection.In contrast,a coinjection(PHAL/CTb)in the ILA reveals that the ILA projects to the mBLAa,but receives input primarily from the lateral BLAa(lBLAa?ILA?mBLAa).Importantly,the retrograde tracer injections in the caudal ILA and PL back-labeled neurons only in the BLAa and in no other amygdalar nuclei highlighting the importance of the BLAa in the MPF-BLA extinction pathway.The mBLAa also sends projections to the dorsal(ACAd)and ventral(ACAv)anterior cingulate cortex and receives minimal input from these cortical areas,the ACA has been shown to process the affective component of nociception,which is an important component of emotional learning,and similar to the PL,the ACAd has been implicated in the expression of conditioned fear.Connections of the orbitofrontal cortex were also examined given its close proximity to the MPF and its involvement in extinction.Experiments demonstrate a role for the orbitofrontal cortex(ORB)in the extinction of aversive olfactory conditioning.The amygdalar inputs to the ORB also appear to originate exclusively in the mBLAa.2.The mBLAa connections to the HPFThe entorhinal cortex(ENT1)is the entryway into the HPF and both the HPF and lateral entorhinal cortex regulate the contextual control of fear extinction.This is a critical sensory input to the network,providing information on environmental context.In fact,lesions of the ENT1 are shown to mimic the deficits of HPF lesions on contextual fear renewal such that animals with a deficient ENTI generalize the safety of one environment to other novel environments.Because extinction is context dependent,the network of regions underlying extinction behavior must include a neural substrate that communicates contextual information.As the mBLAa coinjection shows.the ENT1 inputs to the mBLAa.the SUBv sends input to the mBLAa.but receives projections mainly from the lBLAa.3.Other relevant inputs to the mBLAa.In addition to its connections with the MPF and HPF,the mBLAa communicates with olfactory and thalamic regions possibly involved in extinguishing fears.Olfactory information is the primary sensory modality used by rodents to interact with their environment.is critical for context exploration and recognition.and reasonably comprises an essential part of the circuitry for context-dependent phenomena.Olfactory information is communicated to the mBLAa through its reciprocal connections with the nucleus of the lateral olfactory tract(NLOT)and olfactory processing is influenced by mBLAa unidirectional projections to the posterior ventral anterior olfactory nucleus(AONpv)and to the olfactory tubercle(OT).Other potentially relevant information regarding extinction reaches the mBLAa through the paraventricular thalamic nucleus(PVT).which also shares bidirectional connections with the PL.As such,the PVT serves as an intermediate station for interactions between the mBLAa and MPF.A recent study showed that the PVT is involved in the retrieval of well-consolidated fear memories through its projections to the central amygdalar nucleus.Further.the PVT receives convergent inputs from brain regions in the lower brainstem and hypothalamus that relay information regarding visceral,energy.and behavioral states,and circadian rhythmicity.All of this information relayed to the mBLAa can presumably aid animals to accurately assess environments and to adjust their adaptive behavior based on their homeostatic needs.4 Motor outputs of the networkIt is generally accepted that central amygdala(CEA)projections to the periaqueductal gray(PAG)control the Pavlovianfear response of freezing.The mBLAa PHAL/CTb coinjection shows it to be unidirectionallyconnected to the CEA through which it could directly influence freezing behavior.However.in nature fear drives more complex instrumental behaviors such as fleeing(escape locomotion and jumping).biting.and alarm vocalizations.Therefore.although the autonomic responses to fear are regulated through the BLA-CEA connection,more complex reactions may involve BLA outputs to other motor regions.Accordingly,the mBLA asends unidirectional outputs to the ACAd,the secondary motor cortex(MOs),and to the dorsomedial part of the caudal caudoputamen(CP).The roles of the CP and MOs in planning and coordinating movement are thoroughly demonstrated and the ACAd is speculated to participate in the control of eye movements for scanning environments,which would be important for defensive escape.Once again,of all the amygdalar nuclei,mBLAa neurons are back-labeled when retrograde tracer injections are made into these specific areas ofthe ACAd,MOs,and dorsomedial CP.Hence the mBLAa targets a collection of important cortical and striatal motor areas that could allow it to influence the expression of extinction.In the realm of fear conditioning,the CP has received relatively little attention.This is perhaps because investigations have found that the CP is not critical for the expression of Pavlovian conditioned emotional response of freezing,which has been the primary conditioned response studied in fear.However,the CP appears to form an important part of the extinction network.The PL,which is implicated in the expression of extinction,also projects to this part of the CPdm.Moreover,the part of the PL that projects to CPdm is innervated by the mBLAa.Visuospatial information from the visual cortex(VIS)and retrosplenial area(RSP)is relayed to the CPdm,providing critical sensory input which may be integrated as environmental context to modulate fear expression during extinction or contextual renewal.Together;these strongly suggest that the CP is structurally positioned to participate in fear extinction.On the other hand,the lBLAa-innervated ventrolateralpart of the caudal CP(CPvl)appears to form part of a segregated network.The CPvl receives converging input from the lBLAa,olfactory,viscerosensory,and orofacial somatic inputs from the PIRp,visceral cortex(VISC),and primary somatosensory cortex(SSp)mouth(SSp-m)and nose(SSp-n)divisions,and from the dorsal agranular insular cortex.The PIRp and Aid in turn project to the lBLAa.The convergence of viscerosensory and chemosensory information within the ventrolateral CP could suggest its involvement in ingestive behaviors.In fact,behavioral studies show this part of the CP to be fundamental for orofacial and forelimb motor control of eating and for stereotyped actions of predatory hunting like biting,tongue movements.In general,the mBLAa is an important node in regulating extinction.A final point on the distinct medial and lateral BLAa networks is the finding that the two nuclei do not appear to connect with one another.The PHAL/CTb injection in the mBLAa did not produce any labeling in thelBLAa.Information from the lBLAa could be relayed only indirectly to the mBLAa through the ILA(lBLAa?ILA?mBLAa).This lack of direct communication between the mBLAa and lBLAa emphasizes the segregation of these BLAa subdivisions and can generate.CONCLUSION1)The mBLAa is connected with prefrontal cortical structures implicated in fear extinction like the ILA,PL,ACA,ORB.2)The mBLAa is connected with hippocampal structures like the ENT1 and SUB,areas known for their involvement in environmental context which are likely important for associating an extinction memory.3)The mBLAa connects with olfactory regions like the NLOT,AON,and OT,which are important in providing an olfactory context to extinction memories.4)The mBLAa receives input from thalamic nuclei,like the PVT,which is implicated in the retrieval of fearful memories.5)The mBLAa sends unidirectional input to motor areas like the MOs,CEA,and CP giving it the ability to influence behavioral expressions of fear extinction such as freezing and avoidance. |
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