Anterograde Tracer Phaseolus Vulgaris Leucoagglutinin Research Articles

Effects of ovariectomy on the inputs from the medial nucleus of the amygdala to the ventromedial nucleus of the hypothalamus in young adult rats

During puberty, sexual hormones induce crucial changes in neural circuit organization, leading to significant sexual dimorphism in adult behaviours. The ventrolateral division of the ventromedial nucleus of the hypothalamus (VMHvl) is the major neural site controlling the receptive component of female sexual behaviour, which is dependent on ovarian hormones. The inputs to the VMHvl, originating from the medial nucleus of the amygdala (MeA), transmit essential information to trigger such behaviour. In this study, we investigated the projection pattern of the MeA to the VMHvl in ovariectomized rats at early puberty. Six-week-old Sprague-Dawley rats were ovariectomized (OVX) and, upon reaching 90 days of age, were subjected to iontophoretic injections of the neuronal anterograde tracer Phaseolus vulgaris leucoagglutinin into the MeA. Projections from the MeA to the VMHvl and to other structures included in the neural circuit responsible for female sexual behaviour were analysed in the Control and OVX groups. The results of the semi-quantitative analysis showed that peripubertal ovariectomy reduced the density of intra-amygdalar fibres. The stereological estimates, however, failed to find changes in the organization of the terminal fields of nerve fibres from the MeA to the VMHvl in the adult. The present data show that ovariectomized rats during the peripubertal phase did not undergo significant changes in MeA fibres reaching the VMHvl; however, they suggest a possible effect of ovariectomy on MeA connectivity under amygdalar subnuclei.

Effects of ovariectomy on inputs from the medial preoptic area to the ventromedial nucleus of the hypothalamus of young adult rats.

Puberty is an important phase of development when the neural circuit organization is transformed by sexual hormones, inducing sexual dimorphism in adult behavioural responses. The principal brain area responsible for the control of the receptive component of female sexual behaviour is the ventrolateral division of the ventromedial nucleus of the hypothalamus (VMHvl), which is known for its dependency on ovarian hormones. Inputs to the VMHvl originating from the medial preoptic nucleus (MPN) are responsible for conveying essential information that will trigger such behaviour. Here, we investigated the pattern of the projection of the MPN to the VMHvl in rats ovariectomized at the onset of puberty. Sprague Dawley rats were ovariectomized (OVX) at puberty and then subjected to iontophoretic injections of the neuronal anterograde tracer Phaseolus vulgaris leucoagglutinin into the MPN once they reached 90days of age. This study analysed the connectivity pattern established between the MPN and the VMH that is involved in the neuronal circuit responsible for female sexual behaviour in control and OVX rats. The data show the changes in the organization of the connections observed in the OVX adult rats that displayed a reduced axonal length for the MPN fibres reaching the VMHvl, suggesting that peripubertal ovarian hormones are relevant to the organization of MPN connections with structures involved in the promotion of female sexual behaviour.

The claustrum is a target for projections from the supramammillary nucleus in the rat

Injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) into the rat rostral and caudal supramammillary nucleus (SUM) provided expected patterns of projections into the hippocampus and the septal region. In addition, unexpectedly intense projections were observed into the claustrum defined by parvalbumin expression. Injections of the retrograde tracer fluorogold (FG) into the hippocampus and the region of the claustrum showed that the cells of origin of these projections distributed similarly within the borders of the SUM. The SUM is usually involved in control of hippocampal theta activity, but the observation of intense projections into the claustrum indicates that it may also influence isocortical processes. Therefore, the SUM may coordinate sensory processing in the isocortex with memory formation in the hippocampus.

Differential innervation of superficial versus deep laminae of the dorsal horn by bulbo-spinal serotonergic pathways in the rat

Convergent data showed that bulbo-spinal serotonergic projections exert complex modulatory influences on nociceptive signaling within the dorsal horn. These neurons are located in the B3 area which comprises the median raphe magnus (RMg) and the lateral paragigantocellular reticular (LPGi) nuclei. Because LPGi 5-HT neurons differ from RMg 5-HT neurons regarding both their respective electrophysiological properties and responses to noxious stimuli, we used anatomical approaches for further characterization of the respective spinal projections of LPGi versus RMg 5-HT neuron subgroups.Adult Sprague-Dawley rats were stereotaxically injected into the RMg or the LPGi with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L). The precise location of injection sites and RMg vs LPGi spinal projections into the different dorsal horn laminae were visualized by PHA-L immunolabeling. Double immunofluorescent labeling of PHA-L and the serotonin transporter (5-HTT) allowed detection of serotonergic fibers among bulbo-spinal projections.Anterograde tracing showed that RMg neurons project preferentially into the deep laminae V-VI whereas LPGi neuron projections are confined to the superficial laminae I-II of the ipsilateral dorsal horn. All along the spinal cord, double-labeled PHA-L/5-HTT immunoreactive fibers, which represent only 5–15% of all PHA-L-immunoreactive projections, exhibit the same differential locations depending on their origin in the RMg versus the LPGi.The clear-cut distinction between dorsal horn laminae receiving bulbo-spinal serotonergic projections from the RMg versus the LPGi provides further anatomical support to the idea that the descending serotonergic pathways issued from these two bulbar nuclei might exert different modulatory influences on the spinal relay of pain signaling neuronal pathways.

Cat's medullary reticulospinal and subnucleus reticularis dorsalis noxious neurons form a coupled neural circuit through collaterals of descending axons.

Animals and human beings sense and react to real/potential dangerous stimuli. However, the supraspinal mechanisms relating noxious sensing and nocifensive behavior are mostly unknown. The collateralization and spatial organization of interrelated neurons are important determinants of coordinated network function. Here we electrophysiologically studied medial medullary reticulospinal neurons (mMRF-RSNs) antidromically identified from the cervical cord of anesthetized cats and found that 1) more than 40% (79/183) of the sampled mMRF-RSNs emitted bifurcating axons running within the dorsolateral (DLF) and ventromedial (VMF) ipsilateral fascicles; 2) more than 50% (78/151) of the tested mMRF-RSNs with axons running in the VMF collateralized to the subnucleus reticularis dorsalis (SRD) that also sent ipsilateral descending fibers bifurcating within the DLF and the VMF. This percentage of mMRF collateralization to the SRD increased to more than 81% (53/65) when considering the subpopulation of mMRF-RSNs responsive to noxiously heating the skin; 3) reciprocal monosynaptic excitatory relationships were electrophysiologically demonstrated between noxious sensitive mMRF-RSNs and SRD cells; and 4) injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin evidenced mMRF to SRD and SRD to mMRF projections contacting the soma and proximal dendrites. The data demonstrated a SRD-mMRF network interconnected mainly through collaterals of descending axons running within the VMF, with the subset of noxious sensitive cells forming a reverberating circuit probably amplifying mutual outputs simultaneously regulating motor activity and spinal noxious afferent input. The results provide evidence that noxious stimulation positively engages a reticular SRD-mMRF-SRD network involved in pain-sensory-to-motor transformation and modulation.

Cortical inputs innervate calbindin-immunoreactive interneurons of the rat basolateral amygdaloid complex.

The present study was undertaken to shed light on the synaptic organization of the rat basolateral amygdala (BLA). The BLA contains multiple types of GABAergic interneurons that are differentially connected with extrinsic afferents and other BLA cells. Previously, it was reported that parvalbumin immunoreactive (PV(+) ) interneurons receive strong excitatory inputs from principal BLA cells but very few cortical inputs, implying a prevalent role in feedback inhibition. However, because prior physiological studies indicate that cortical afferents do trigger feedforward inhibition in principal cells, the present study aimed to determine whether a numerically important subtype of interneurons, expressing calbindin (CB(+) ), receives cortical inputs. Rats received injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) in the perirhinal cortex or adjacent temporal neocortex. Light and electron microscopic observations of the relations between cortical inputs and BLA neurons were performed in the lateral (LA) and basolateral (BL) nuclei. Irrespective of the injection site (perirhinal or temporal neocortex) and target nucleus (LA or BL), ~90% of cortical axon terminals formed asymmetric synapses with dendritic spines of principal BLA neurons, while 10% contacted the dendritic shafts of presumed interneurons, half of which were CB(+) . Given the previously reported pattern of CB coexpression among GABAergic interneurons of the BLA, these results suggest that a subset of PV-immunonegative cells that express CB, most likely the somatostatin-positive interneurons, are important mediators of cortically evoked feedforward inhibition in the BLA.

Lateral habenula and the rostromedial tegmental nucleus innervate neurochemically distinct subdivisions of the dorsal raphe nucleus in the rat

The lateral habenula (LHb) is an epithalamic structure differentiated in a medial (LHbM) and a lateral division (LHbL). Together with the rostromedial tegmental nucleus (RMTg), the LHb has been implicated in the processing of aversive stimuli and inhibitory control of monoamine nuclei. The inhibitory LHb influence on midbrain dopamine neurons has been shown to be mainly mediated by the RMTg, a mostly GABAergic nucleus that receives a dominant input from the LHbL. Interestingly, the RMTg also projects to the dorsal raphe nucleus (DR), which also receives direct LHb projections. To compare the organization and transmitter phenotype of LHb projections to the DR, direct and indirect via the RMTg, we first placed injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin into the LHb or the RMTg. We then confirmed our findings by retrograde tracing and investigated a possible GABAergic phenotype of DR-projecting RMTg neurons by combining retrograde tracing with in situ hybridization for GAD67. We found only moderate direct LHb projections to the DR, which mainly emerged from the LHbM and were predominantly directed to the serotonin-rich caudal DR. In contrast, RMTg projections to the DR were more robust, emerged from RMTg neurons enriched in GAD67 mRNA, and were focally directed to a distinctive DR subdivision immunohistochemically characterized as poor in serotonin and enriched in presumptive glutamatergic neurons. Thus, besides its well-acknowledged role as a GABAergic control center for the ventral tegmental area (VTA)-nigra complex, our findings indicate that the RMTg is also a major GABAergic relay between the LHb and the DR.

Lateral habenula and the rostromedial tegmental nucleus innervate neurochemically distinct subdivisions of the dorsal raphe nucleus in the rat

ABSTRACTThe lateral habenula (LHb) is an epithalamic structure differentiated in a medial (LHbM) and a lateral division (LHbL). Together with the rostromedial tegmental nucleus (RMTg), the LHb has been implicated in the processing of aversive stimuli and inhibitory control of monoamine nuclei. The inhibitory LHb influence on midbrain dopamine neurons has been shown to be mainly mediated by the RMTg, a mostly GABAergic nucleus that receives a dominant input from the LHbL. Interestingly, the RMTg also projects to the dorsal raphe nucleus (DR), which also receives direct LHb projections. To compare the organization and transmitter phenotype of LHb projections to the DR, direct and indirect via the RMTg, we first placed injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin into the LHb or the RMTg. We then confirmed our findings by retrograde tracing and investigated a possible GABAergic phenotype of DR‐projecting RMTg neurons by combining retrograde tracing with in situ hybridization for GAD67. We found only moderate direct LHb projections to the DR, which mainly emerged from the LHbM and were predominantly directed to the serotonin‐rich caudal DR. In contrast, RMTg projections to the DR were more robust, emerged from RMTg neurons enriched in GAD67 mRNA, and were focally directed to a distinctive DR subdivision immunohistochemically characterized as poor in serotonin and enriched in presumptive glutamatergic neurons. Thus, besides its well‐acknowledged role as a GABAergic control center for the ventral tegmental area (VTA)–nigra complex, our findings indicate that the RMTg is also a major GABAergic relay between the LHb and the DR. J. Comp. Neurol. 522:1454–1484, 2014. © 2013 Wiley Periodicals, Inc.

Oxytocin projections to the nucleus of the solitary tract contribute to the increased meal-related satiety responses in primary adrenal insufficiency.

Anorexia is a common clinical manifestation of primary adrenal gland failure. Adrenalectomy (ADX)-induced hypophagia is reversed by oxytocin (OT) receptor antagonist and is associated with increased activation of satiety-related responses in the nucleus of the solitary tract (NTS). This study evaluated OT projections from the paraventricular nucleus of the hypothalamus (PVN) to the NTS after ADX and the effect of pretreatment with intracerebroventricular injection of an OT receptor antagonist ([d(CH2)5,Tyr(Me)(2),Orn(8)]-vasotocin; OVT) on the activation of NTS neurons induced by feeding in adrenalectomized rats. Adrenalectomized animals showed higher OT labelling in the NTS than the sham and the ADX with corticosterone replacement (ADX+B) groups. Adrenalectomized animals exhibited co-localization of the anterograde tracer Phaseolus vulgaris leucoagglutinin and OT in axons in the NTS as well as OT fibres apposing NTS neurons activated by refeeding. After vehicle pretreatment, compared with fasting, refeeding increased the numbers of Fos- and Fos+TH-immunoreactive neurons in the NTS in sham, ADX and ADX+B groups, with a higher number of these immunolabelled neurons in adrenalectomized animals. Compared with fasting conditions, refeeding also increased the activation of NTS neurons in OVT-pretreated sham, ADX and ADX+B groups, but there was no difference among the three experimental groups. These data demonstrate that OT is upregulated in projections to the NTS following ADX and that OT receptor antagonist reverses the greater activation of NTS neurons induced by feeding after ADX. The data indicate that OT pathways to the NTS contribute to higher satiety-related responses and, thus, to reduce meal size in primary adrenal insufficiency.

Differential connectivity of short- vs. long-range extrinsic and intrinsic cortical inputs to perirhinal neurons.

The perirhinal cortex plays a critical role in recognition and associative memory. However, the network properties that support perirhinal contributions to memory are unclear. To shed light on this question, we compared the synaptic articulation of short- and long-range inputs from the perirhinal cortex or temporal neocortex with perirhinal neurons in rats. Iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) were performed at different rostrocaudal levels of the ventral temporal neocortex or perirhinal cortex, and electron microscopic observations of anterogradely labeled (PHAL(+)) axon terminals found at perirhinal sites adjacent to or rostrocaudally distant from the injection sites were performed. After neocortical injections, the density of PHAL(+) axons in the perirhinal cortex decreased steeply with rostrocaudal distance from the injection sites, much more so than following perirhinal injections. Otherwise, similar results were obtained with neocortical and perirhinal injections. In both cases, most (76-86%) PHAL(+) axon terminals formed asymmetric synapses, typically with spines (type A, 83-89%) and less frequently with dendritic profiles (type B, 11-17%). The remaining terminals formed symmetric synapses with dendritic profiles (type C, 14-23%). Type B and C synapses were 2.4-2.6 times more frequent in short- than long-range connections. The postsynaptic elements in type A-C synapses were identified with immunocytochemistry for CAMKIIα, a marker of glutamatergic cortical neurons. Type A and C terminals contacted CAMKIIα-positive principal cells, whereas type B synapses contacted presumed inhibitory neurons. Overall, these results suggest that principal perirhinal neurons are subjected to significantly more inhibition from short- than from long-range cortical inputs, an organization that likely impacts perirhinal contributions to memory.

Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat

The mesopontine rostromedial tegmental nucleus (RMTg) is a mostly γ-aminobutyric acid (GABA)ergic structure believed to be a node for signaling aversive events to dopamine (DA) neurons in the ventral tegmental area (VTA). The RMTg receives glutamatergic inputs from the lateral habenula (LHb) and sends substantial GABAergic projections to the VTA, which also receives direct projections from the LHb. To further specify the topography of LHb projections to the RMTg and VTA, small focal injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin were aimed at different subdivisions of the LHb. The subnuclear origin of LHb inputs to the VTA and RMTg was then confirmed by injections of the retrograde tracer cholera toxin subunit b into the VTA or RMTg. Furthermore, we compared the topographic position of retrogradely labeled neurons in the RMTg resulting from VTA injections with that of anterogradely labeled axons emerging from the LHb. As revealed by anterograde and retrograde tracing, LHb projections were organized in a strikingly topographic manner, with inputs to the RMTg mostly arising from the lateral division of the LHb (LHbL), whereas inputs to the VTA mainly emerged from the medial division of the LHb (LHbM). In the RMTg, profusely branched LHb axons were found in close register with VTA projecting neurons and were frequently apposed to the latter. Overall, our findings demonstrate that LHb inputs to the RMTg and VTA arise from different divisions of the LHb and provide direct evidence for a disynaptic pathway that links the LHbL to the VTA via the RMTg.

Projections of the medial orbital and ventral orbital cortex in the rat

The medial orbital (MO) and ventral orbital (VO) cortices are prominent divisions of the orbitomedial prefrontal cortex. To our knowledge, no previous report in the rat has comprehensively described the projections of MO and VO. By using the anterograde tracer Phaseolus vulgaris leucoagglutinin and the retrograde tracer Fluoro-Gold, we examined the efferent projections of MO and VO in the rat. Although MO and VO projections overlap, MO distributes more widely throughout the brain, particularly to limbic structures, than does VO. The main cortical targets of MO were the orbital, ventral medial prefrontal (mPFC), agranular insular, piriform, retrosplenial, and parahippocampal cortices. The main subcortical targets of MO were the medial striatum, olfactory tubercle, claustrum, nucleus accumbens, septum, substantia innominata, lateral preoptic area, and diagonal band nuclei of the basal forebrain; central, medial, cortical, and basal nuclei of amygdala; paratenial, mediodorsal, and reuniens nuclei of the thalamus; posterior, supramammillary, and lateral nuclei of the hypothalamus; and periaqueductal gray, ventral tegmental area, substantia nigra, dorsal and median raphe, laterodorsal tegmental, and incertus nuclei of the brainstem. By comparison, VO distributes to some of these same sites, notably to the striatum, but lacks projections to parts of limbic cortex, to nucleus accumbens, and to the amygdala. VO distributes much more strongly, however, than MO to the medial (frontal) agranular, anterior cingulate, sensorimotor, posterior parietal, lateral agranular retrosplenial, and temporal association cortices. The patterns of MO projections are similar to those of the mPFC, whereas the projections of VO overlap with those of the ventrolateral orbital cortex (VLO). This suggests that MO serves functions comparable to those of the mPFC, such as goal-directed behavior, and VO performs functions similar to VLO such as directed attention. MO/VO may also serve as a link between lateral orbital and medial prefrontal cortices.

Synaptology of ventral CA1 and subiculum projections to the basomedial nucleus of the amygdala in the mouse: relation to GABAergic interneurons

GABAergic neurons of the amygdala are thought to play a critical role in establishing networks for feedback and feedforward inhibition and in mediating rhythmic network activity patterns relevant for emotional behavior, determination of stimulus salience, and memory strength under stressful experiences. These functions are typically fulfilled in interplay of amygdala and hippocampus. Therefore, we explored the putative connectivity of GABAergic neurons with the hippocampo-amygdalar projection with the anterograde tracers Phaseolus vulgaris leucoagglutinin (Phal) and Miniruby injected to GAD67-GFP knock-in mice in which GABAergic neurons are labeled by the expression of the gene for green fluorescent protein (GFP) inserted to the GAD1 gene locus (Tamamaki et al. J Comp Neurol 467:60-79, 2003). We found that, while hippocampal axons target all nuclei of the amygdala, the densest fiber plexus was found in the posterior basomedial nucleus. Electron microscopy revealed that the vast majority of contacts in this nucleus were formed by thin fibers making small asymmetrical contacts, predominantly on GFP-negative profiles. However, several asymmetrical contacts could also be seen on GFP-positive profiles. A surprising result was the occasional occurrence of anterogradely labeled symmetrical synapses indicating a GABAergic contribution to the projection from the hippocampus to the amygdala. While hippocampal input to the amygdala appears to be largely excitatory and targets non-GABAergic neurons, our data provide evidence for a direct involvement of GABAergic neurons in the interplay of these regions, either as target in the amygdala or as projection neurons from the hippocampus. These particular "interface neurons" may be of relevance for the information processing in the amygdalo-hippocampal system involved in emotional behavior and memory formation.

Organization of projections from the spinal trigeminal subnucleus oralis to the spinal cord in the rat: A neuroanatomical substrate for reciprocal orofacial–cervical interactions

The organization of efferent projections from the spinal trigeminal nucleus oralis (Sp5O) to the spinal cord in the rat was studied using the anterograde tracer Phaseolus vulgaris leucoagglutinin. Sp5O projections to the spinal cord are restricted to the cervical cord. No labeled terminal can be detected in the thoracic and lumbar cord. The organization of these projections happens to critically depend on the dorso-ventral location of the injection site. On the one hand, the dorsal part of the Sp5O projects to the medial part of the dorsal horn (laminae III–V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the ipsilateral one. Ipsilateral labeled terminals are distributed throughout laminae VII to IX but tend to cluster around the dorso-medial motor nuclei, especially at C3–C5 levels. Within the contralateral ventral horn, label terminals are found particularly in the region of the ventro-medial motor nucleus. This projection extends as far caudally as C3 or C4 level. On the other hand, the ventral part of the Sp5O projects to the lateral part of the dorsal horn (laminae III–V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the contralateral one. Contralateral labeled terminals are distributed within the region of the dorso- and ventro-medial motor nuclei at C1–C4 levels whereas they are restricted to the dorso-medial motor nucleus at C5–C8 levels. These findings suggest that Sp5O is involved in the coordination of neck movements and in the modulation of incoming sensory information at the cervical spinal cord.

Projections from the inferior colliculus to the tectal longitudinal column in the rat

We have used the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) to study with albino rats the projections from the inferior colliculus (IC) to the tectal longitudinal column (TLC), a newly discovered nucleus that spans the midbrain tectum longitudinally, on each side of the midbrain, immediately above the periaqueductal gray matter. We studied the projections of the medial IC, which includes the classical central nucleus (CNIC) and the dorsal cortex (DCIC), and those of the lateral IC, equivalent to the classical external cortex (ECIC). Following unilateral injections of PHA-L into the medial IC, numerous terminal fibers are labeled bilaterally in the TLC. The ipsilateral projection is denser and targets the entire nucleus, whereas the contralateral projection targets significantly only the caudal half or two-thirds of the TLC. Fibers from the medial IC reach the TLC by two routes: as collaterals of axons that travel in the commissure of the IC and as collaterals of thick ipsilateral colliculogeniculate axons; the latter travel through the deep superior colliculus on their way to the TLC. Within the TLC, individual IC fibers tend to run longitudinally. The injection of PHA-L into the lateral IC indicates that this subdivision sends a weak, bilateral projection to the TLC whose trajectory, morphology and distribution are similar to those of the projection from the medial IC. These results demonstrate that all subdivisions of the IC send projections to the TLC, suggesting that the IC may be one of the main sources of auditory input to this tectal nucleus.

Ultrastructural analysis of local collaterals of rat ventral tegmental area neurons: GABA phenotype and synapses onto dopamine and GABA cells

Local synapses formed by nondopamine cells within the ventral tegmental area (VTA) are thought to provide an important regulatory influence on the activity patterns of dopamine (DA) neurons. However, ultrastructural confirmation of intra-areal synapses formed by VTA neurons is lacking, and the synaptic targets of these connections have not been examined. We performed discrete injections of the specific anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) and used electron microscopy to visualize immunoperoxidase labeling within the local collaterals of VTA cells. The phenotype of target neurons was determined by immunogold-silver labeling for GABA or for tyrosine hydroxylase within DA neurons. Within or immediately adjacent to the VTA injection sites, PHAL was incorporated into the soma and dendrites of both GABA and DA cells. Tracer was also detected within myelinated and unmyelinated axons as well as axon terminals. Some labeled terminals formed identifiable synapses, the majority of which (78%) had symmetric morphology (presumably inhibitory). Both DA and GABA dendrites were contacted by these intrinsic axons. Postembedding immunogold labeling verified that local axon collaterals arose mainly from GABA cells (DA neurons are not known to issue recurrent collaterals). Nevertheless, a few synapses with asymmetric morphology (presumably excitatory) were also noted; whether these derive from local glutamate neurons requires further investigation. Hence, our data provide ultrastructural support for the long standing assumption that GABA VTA neurons synapse locally onto DA cells. The findings also suggest the presence of disinhibitory and possibly excitatory circuitry intrinsic to the VTA.

Efferent projections of thyrotropin‐releasing hormone‐synthesizing neurons residing in the anterior parvocellular subdivision of the hypothalamic paraventricular nucleus

The anterior parvocellular subdivision of the PVN (aPVN) contains nonhypophysiotropic thyrotropin-releasing hormone (TRH) neurons that are densely innervated by feeding-related neuronal groups of the hypothalamic arcuate nucleus. To determine how these TRH neurons are integrated within the brain, the major projection fields of this cell group were studied by anterograde and retrograde tract-tracing methods. Projection sites were identified by injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) into the aPVN, and subsequent double immunofluorescent staining was used to visualize axons containing both PHAL and pro-TRH. To distinguish between the projection sites of TRH neurons residing in the aPVN and the closely situated perifornical area, the retrograde tracer cholera toxin B subunit (CTB) was injected into regions where PHAL/pro-TRH-containing axons were densely accumulated. TRH neurons in the aPVN were found to project to the hypothalamic arcuate, dorsomedial and ventral premammillary nuclei, medial preoptic region, tuber cinereum area, paraventricular thalamic nucleus, bed nucleus of the stria terminalis, lateral septal nucleus, and central amygdaloid nucleus. Projection fields of perifornical TRH neurons were in partial overlap with those of the aPVN TRH cells. In addition, these neurons also innervated the hypothalamic ventromedial nucleus, the medial amygdaloid nucleus, and the amygdalohippocampal area. The data suggest that, through its efferent connections, aPVN TRH neurons may be involved in the regulation of energy homeostasis coordinately with effects on behavior, locomotor activity, and thermogenesis. In addition, the major differences in the projection fields of aPVN and perifornical TRH neurons suggest that these two TRH-synthesizing neuronal groups are functionally different.

Contralateral projections of the rat anterior olfactory nucleus.

The anterior olfactory nucleus (AON) is a central olfactory cortical structure that has heavy reciprocal connections with both the olfactory bulb (OB) and piriform cortex. While it has been firmly established that the AON is a primary source of bilateral projections in the olfactory system through extensive connections with both the ipsilateral and contralateral OB, AON, and piriform cortex, few studies have examined this circuitry in detail. In the present study we used small injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde tracer FluoroGold in specific subregions of the AON to explore the topography of the interconnections between the left and right AONs. Labeled fibers were found in the contralateral AON following injections in all areas. However, detailed quantitative analyses revealed that different regions of the AON have distinct patterns of interhemispheric innervation; contralateral fibers were most heavily targeted to dorsal and lateral AON subregions, while the medial and ventral areas received relatively light projections. These results demonstrate important features of the interhemispheric circuitry of the AON and suggest separate functional roles for subregions of the AON in olfactory information processing.

Organization of connectivity of the rat presubiculum: II. Associational and commissural connections

The regional, laminar, and longitudinal organization of intrinsic projections in the presubiculum was examined in the rat with the retrograde tracer horseradish peroxidase conjugated to wheat germ agglutinin and the anterograde tracer Phaseolus vulgaris-leucoagglutinin. Cells of origin of intrinsic projections in the presubiculum were distributed in layers II and V, with almost none in layers III and VI. Projections from layer II cells were bilateral and confined to layers II and V and also to the deep portion of layer I, whereas projections from layer V cells were ipsilateral and confined largely to layer V, with fewer projections to layer II. Septotemporal and proximodistal differences in both the projection and the distribution of layer II cells were found: layer II cells in the septal and mid presubiculum, especially those located in the distal part, provided long projections to the temporal presubiculum, whereas layer II cells in the temporal presubiculum provided slightly shorter projections almost entirely within the mid and temporal presubiculum. Layer II cells aggregated massively in the distal portions of the septal and mid presubiculum, but very few layer II cells were found in the most proximal part, especially in the temporal presubiculum. On the other hand, in layer V, cells of origin and their terminals were diffusely and equally distributed throughout the entire proximodistal extent of the presubiculum. Layer V cells did not project longitudinally as far as layer II cells. These longitudinal connections, in layers II and V, make it possible to merge information conveyed by parallel pathways in the presubiculum.

Cytoarchitectonics and afferent/efferent reorganization of neurons in layers II and III of the lateral entorhinal cortex in the mouse pilocarpine model of temporal lobe epilepsy

With the mouse pilocarpine model of temporal lobe epilepsy (TLE), we showed a progressive loss of both principal cells and calbindin (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunopositive interneurons in layers II-III of lateral entorhinal cortex (LEnt) from 2 months to 1 year after pilocarpine-induced status epilepticus (PISE). In the efferent pathway of LEnt, more Phaseolus vulgaris leucoagglutinin (PHA-L)-labelled en passant and terminal boutons with larger diameters were shown in the hippocampus and subiculum; in the prefrontal, piriform, and perirhinal cortices; and in the amygdaloid complex in experimental mice at the two time points compared with the control after iontophoretical injection of an anterograde tracer PHA-L into the LEnt. Furthermore, the numbers of CB- or CR-immunopositive neurons contacted by PHA-L-labelled en passant and terminal boutons decreased in most of these areas at 2 months or 1 year after PISE. In the afferent pathway of LEnt, the numbers of retrogradely labelled neurons were reduced significantly in the ipsilateral piriform cortex and endopiriform nucleus at 2 months and 1 year and in the reuniens thalamic nucleus only at 1 year after injection of a retrograde tracer cholera toxin B subunit (CTB) into the LEnt. The percentages of the number of CTB and CB or CR double-labelled neurons of all the retrogradely labelled neurons were also decreased in the reunions thalamic nucleus at 1 year after PISE. It is concluded that both cytoarchitectonic change and reorganization of afferent and efferent pathways in LEnt may be involved in the occurrence of TLE.