Presynaptic kainate receptors at hippocampal mossy fiber synapses

Hippocampal mossy fibers, which are the axons of dentate granule cells, form powerful excitatory synapses onto the proximal dendrites of CA3 pyramidal cells. It has long been known that high-affinity binding sites for kainate, a glutamate receptor agonist, are present on mossy fibers. Here we summarize recent experiments on the role of these presynaptic kainate receptors (KARs). Application of kainate has a direct effect on the amplitude of the extracellularly recorded fiber volley, with an enhancement by low concentrations and a depression by high concentrations. These effects are mediated by KARs, because they persist in the presence of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-selective antagonist GYKI 53655, but are blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/KAR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and the KAR antagonist SYM2081. The effects on the fiber volley are most likely caused by a depolarization of the fibers via the known ionotropic actions of KARs, because application of potassium mimics the effects. In addition to these effects on fiber excitability, low concentrations of kainate enhance transmitter release, whereas high concentrations depress transmitter release. Importantly, the synaptic release of glutamate from mossy fibers also activates these presynaptic KARs, causing an enhancement of the fiber volley and a facilitation of release that lasts for many seconds. This positive feedback contributes to the dramatic frequency facilitation that is characteristic of mossy fiber synapses. It will be interesting to determine how widespread facilitatory presynaptic KARs are at other synapses in the central nervous system.     

Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses

The release properties of synapses in the central nervous system vary greatly, not only across anatomically distinct types of synapses but also among the same class of synapse. This variation manifests itself in large part by differences in the probability of transmitter release, which affects such activity-dependent presynaptic forms of plasticity as paired-pulse facilitation and frequency facilitation. This heterogeneity in presynaptic function reflects differences in the intrinsic properties of the synaptic terminal and the activation of presynaptic neurotransmitter receptors. Here we show that the unique presynaptic properties of the hippocampal mossy fiber synapse are largely imparted onto the synapse by the continuous local action of extracellular adenosine at presynaptic A1 adenosine receptors, which maintains a low basal probability of transmitter release.     

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.     

Perinatal hypothyroidism decreases hippocampal mossy fiber zinc density in rats.

The effect of perinatal hypothyroidism on hippocampal mossy fiber zinc density was examined in rats. Timed pregnant Sprague-Dawley rat dams were given water containing either 0.02% propylthiouracil (PTU) or vehicle from gestational day 18 until their litters were weaned on postnatal day 31. Hippocampal mossy fiber zinc density was reduced by 75% in both the dorsal and ventral hippocampal formation CA3 stratum lucidum region of 31-day-old PTU-treated rats compared to untreated controls. Perinatal hypothyroidism did not alter hippocampal tissue zinc concentration, indicating that the PTU-induced reduction in mossy fiber zinc was not a consequence of reduced hippocampal zinc concentration. At 120 days of age, 3 months after discontinuation of PTU treatment, hippocampal mossy fiber zinc density remained significantly reduced by 33-45% in PTU-treated rats compared to control. These data indicate that perinatal hypothyroidism causes a long-lasting reduction in hippocampal mossy fiber zinc density.     

Development of hippocampal mossy fiber synaptic outputs by new neurons in the adult brain

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.     

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.     

GluR7 is an essential subunit of presynaptic kainate autoreceptors at hippocampal mossy fiber synapses

Presynaptic ionotropic glutamate receptors are emerging as key players in the regulation of synaptic transmission. Here we identify GluR7, a kainate receptor (KAR) subunit with no known function in the brain, as an essential subunit of presynaptic autoreceptors that facilitate hippocampal mossy fiber synaptic transmission. GluR7(-/-) mice display markedly reduced short- and long-term synaptic potentiation. Our data suggest that presynaptic KARs are GluR6/GluR7 heteromers that coassemble and are localized within synapses. We show that recombinant GluR6/GluR7 KARs exhibit low sensitivity to glutamate, and we provide evidence that presynaptic KARs at mossy fiber synapses are likely activated by high concentrations of glutamate. Overall, from our data, we propose a model whereby presynaptic KARs are localized in the presynaptic active zone close to release sites, display low affinity for glutamate, are likely Ca(2+)-permeable, are activated by single release events, and operate within a short time window to facilitate the subsequent release of glutamate.     

Two-photon imaging of Zn2+ dynamics in mossy fiber boutons of adult hippocampal slices

Mossy fiber termini in the hippocampus accumulate Zn²⁺, which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn²⁺ at the synaptic level is challenging owing to the difficulty of visualizing Zn²⁺ at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn²⁺ dynamics mediated by NMDA receptor-dependent long-term potentiation induction at single mossy fiber termini of dentate gyrus neurons in adult mouse hippocampal slices. The membrane-impermeant fluorescent Zn²⁺ probe, 6-CO₂H-ZAP4, was loaded into presynaptic vesicles in hippocampal mossy fiber termini upon KCl-induced depolarization, which triggers subsequent endocytosis and vesicular restoration. Local tetanic stimulation decreased the Zn²⁺ signal observed at individual presynaptic sites, indicating release of the Zn²⁺ from vesicles in synaptic potentiation. This synapse-level two-photon Zn²⁺ imaging method enables monitoring of presynaptic Zn²⁺ dynamics for improving the understanding of physiological roles of mobile Zn²⁺ in regular and aberrant neurologic function.Although most cellular Zn²⁺ is sequestered within proteins, stores of loosely bound Zn²⁺ are present in many kinds of cells. This mobile Zn²⁺ pool is believed to mediate cellular processes, including neurotransmission (1). Within the hippocampus, mossy fibers connecting the dentate gyrus (DG) and the CA3 regions contain Zn²⁺ in glutamatergic synaptic vesicles. The precise concentration of Zn²⁺ within the neuronal vesicles is unknown, with upper estimates ranging in the low millimolar range (2, 3). Upon stimulation, Zn²⁺ is believed to be coreleased with glutamate and to modulate glutamatergic synaptic transmission (4, 5). Despite this recent finding, however, much remains to be understood about the dynamics and functional roles of synaptic Zn²⁺.Numerous fluorescent Zn²⁺ probes have been developed for use in biological systems (6, 7), including 6-methoxy-(8-p-toluenesulfonamido)quinoline (8), Zinquin (9), Zinbo-5 (10), and the Zinpyr (ZP) and ZnAF families of probes (11–14). Despite the abundance of fluorescent Zn²⁺ probes, analysis of Zn²⁺ in vivo remains problematic. Many probes bind Zn²⁺ to form complexes with dissociation constants in the nanomolar range; these tight binding affinities lead to rates of Zn²⁺ release that are too slow for time-resolvable measurements. These probes also may act as Zn²⁺ traps, sequestering Zn²⁺ in one region of the cell, and then collecting elsewhere to yield a faulty image of native Zn²⁺ distributions within cells. Reductions in binding affinity can be accomplished via two strategies. First, steric bulk can be installed near the chelating atoms, as was done with two series of methylated Zn²⁺ probes (15, 16). Second, chelating atoms can be removed from the ligand systematically, as was done for probes in the ZnAF series (16), as well as those in the QZ, Zinspy, and ZinAlkylPyr (ZAP) families (17–19).Here we present the synthesis of the probe ZinAlkylPyr-4 (ZAP4) and its 6-carboxylic acid derivative (6-CO₂H-ZAP4). The molecular structure of ZAP4 was modeled after earlier ZAP probes (17), with pentafluorobenzyl groups chosen as the alkyl groups to reduce the basicity of the probe and minimize proton-induced enhancement of the emission. The 6-CO₂H-ZAP4 probe was constructed as a membrane-impermeant Zn²⁺ probe, analogous to the previously reported 6-CO₂H-ZP1 (20).Previous attempts to monitor presynaptic Zn²⁺ dynamics in living brain tissues used membrane-permeant probes to detect intracellular Zn²⁺ from populations of presynaptic terminals at relatively low spatial resolution at the tissue level (21, 22). In addition, activity-dependent extracellular Zn²⁺ release has been monitored with membrane-impermeant probes (16, 23–30). These tissue level-averaged Zn²⁺ imaging methods have limited spatial and temporal resolution and fail to specify the precise location of the relevant synapses.We describe intracellular Zn²⁺ imaging at the single-synapse level in mossy fiber termini of neurons in acute hippocampal slices from adult mice. The technique relies on the membrane-impermeant fluorescent Zn²⁺ probe 6-CO₂H-ZAP4 and two-photon fluorescence microscopy.     

Use-dependent amplification of presynaptic Ca2+ signaling by axonal ryanodine receptors at the hippocampal mossy fiber synapse

Presynaptic Ca²⁺ stores have been suggested to regulate Ca²⁺ dynamics within the nerve terminals at certain types of the synapse. However, little is known about their mode of activation, molecular identity, and detailed subcellular localization. Here, we show that the ryanodine-sensitive stores exist in axons and amplify presynaptic Ca²⁺ accumulation at the hippocampal mossy fiber synapses, which display robust presynaptic forms of plasticity. Caffeine, a potent drug inducing Ca²⁺ release from ryanodine-sensitive stores, causes elevation of presynaptic Ca²⁺ levels and enhancement of transmitter release from the mossy fiber terminals. The blockers of ryanodine receptors, TMB-8 or ryanodine, reduce presynaptic Ca²⁺ transients elicited by repetitive stimuli of mossy fibers but do not affect those evoked by single shocks, suggesting that ryanodine receptors amplify presynaptic Ca²⁺ dynamics in an activity dependent manner. Furthermore, we generated the specific antibody against the type 2 ryanodine receptor (RyR2; originally referred to as the cardiac type) and examined the cellular and subcellular localization using immunohistochemistry. RyR2 is highly expressed in the stratum lucidum of the CA3 region and mostly colocalizes with axonal marker NF160 but not with terminal marker VGLUT1. Immunoelectron microscopy revealed that RyR2 is distributed around smooth ER within the mossy fibers but is almost excluded from their terminal portions. These results suggest that axonal localization of RyR2 at sites distant from the active zones enables use dependent Ca²⁺ release from intracellular stores within the mossy fibers and thereby facilitates robust presynaptic forms of plasticity at the mossy fiber-CA3 synapse.     

Rapid Ca2+ channel accumulation contributes to cAMP-mediated increase in transmission at hippocampal mossy fiber synapses

The cyclic adenosine monophosphate (cAMP)-dependent potentiation of neurotransmitter release is important for higher brain functions such as learning and memory. To reveal the underlying mechanisms, we applied paired pre- and postsynaptic recordings from hippocampal mossy fiber-CA3 synapses. Ca²⁺ uncaging experiments did not reveal changes in the intracellular Ca²⁺ sensitivity for transmitter release by cAMP, but suggested an increase in the local Ca²⁺ concentration at the release site, which was much lower than that of other synapses before potentiation. Total internal reflection fluorescence (TIRF) microscopy indicated a clear increase in the local Ca²⁺ concentration at the release site within 5 to 10 min, suggesting that the increase in local Ca²⁺ is explained by the simple mechanism of rapid Ca²⁺ channel accumulation. Consistently, two-dimensional time-gated stimulated emission depletion microscopy (gSTED) microscopy showed an increase in the P/Q-type Ca²⁺ channel cluster size near the release sites. Taken together, this study suggests a potential mechanism for the cAMP-dependent increase in transmission at hippocampal mossy fiber-CA3 synapses, namely an accumulation of active zone Ca²⁺ channels.

Communication between neurons is largely mediated by chemical synapses. Synaptic strengths are not fixed, but change dynamically in the short and longer term in an activity-dependent manner (short- and long-term plasticity, 1–3). Moreover, neuromodulators act on presynaptic terminals to modulate synaptic strength. Such activity-dependent or modulatory changes are often mediated by the activation of second messengers, such as protein kinase A and C (2). Second messenger systems, particularly the cyclic adenosine monophosphate (cAMP)/PKA-dependent system, are important for higher brain functions, including learning and memory in Aplysia (3), flies (4, 5), and the mammalian brain (6). Despite its functional importance, the cellular and molecular mechanisms of cAMP-dependent modulation are still poorly understood regardless of whether Aplysia synapses and Drosophila neuromuscular junctions have been investigated (2, 7). Mammalian central synapses are no exception here, also reflecting technical difficulties due to the generally small size of the presynaptic terminals in the mammalian brain.Hippocampal mossy fiber-CA3 (MF-CA3) synapses are characterized by exceptionally large presynaptic terminals (hippocampal mossy fiber bouton, hMFB), which allow for the direct analysis of the cellular mechanisms of synaptic transmission and plasticity by using patch-clamp recordings (8–10). Thus, hMFBs provide a suitable model of cortical synapses in the mammalian brain. Moreover, these synapses are functionally important for brain function such as pattern separation (11). Mossy fiber synapses are known to exhibit unique presynaptic forms of short- and long-term synaptic potentiation and depression, which share the cAMP/PKA-dependent induction mechanism (12–15). In addition, the cAMP-dependent plasticity pathway is important for presynaptic modulation by dopamine and noradrenaline (16–18), which modulates hippocampal network activity and behavior. However, its underlying cellular mechanisms remain largely unclear. Enhancement of the molecular priming and docking of synaptic vesicles at mossy fiber synapses has been suggested by previous studies using genetics and electron microscopy (19–21). In particular, RIM1, an active zone scaffold protein, is crucial for cAMP-dependent long-term potentiation (LTP) (19) and is phosphorylated by PKA, although a corresponding phosphorylation mutant of RIM1 was found to have no effect on long-term potentiation (22, but see ref. 23). Other studies on hMFBs have implicated a role in positional priming, i.e., changes in the spatial coupling between Ca²⁺ channels and the release machinery (24). However, there is a lack of the direct visualization or manipulation of this regulation.In order to measure the intracellular Ca²⁺ sensitivity of transmitter release directly and examine the mechanisms of cAMP-dependent modulation quantitatively, we here carried out Ca²⁺ uncaging experiments at hippocampal mossy fiber synapses. Unexpectedly, our results failed to show changes in Ca²⁺ sensitivity, but instead uncovered an increase in local Ca²⁺ concentrations at the release sites. Furthermore, by live imaging of local Ca²⁺ using total internal reflection fluorescence (TIRF) microscopy as well as superresolution time gated STED (gSTED) microscopy, we provided evidence that rather rapid Ca²⁺ channel accumulation may underlie cAMP-induced potentiation instead of release machinery modulations. This study thus provides a potential mechanism of presynaptic modulation at central synapses.     

Gap junctions on hippocampal mossy fiber axons demonstrated by thin-section electron microscopy and freeze fracture replica immunogold labeling

Gap junctions have been postulated to exist between the axons of excitatory cortical neurons based on electrophysiological, modeling, and dye-coupling data. Here, we provide ultrastructural evidence for axoaxonic gap junctions in dentate granule cells. Using combined confocal laser scanning microscopy, thin-section transmission electron microscopy, and grid-mapped freeze-fracture replica immunogold labeling, 10 close appositions revealing axoaxonic gap junctions ( approximately 30-70 nm in diameter) were found between pairs of mossy fiber axons ( approximately 100-200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction ( approximately 100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus. Immunogold labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data support computer modeling and in vitro electrophysiological data suggesting that axoaxonic gap junctions play an important role in the generation of very fast (>70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy.     

PTP and LTP at a hippocampal mossy fiber-interneuron synapse

The mossy fiber-CA3 pyramidal neuron synapse is a main component of the hippocampal trisynaptic circuitry. Recent studies, however, suggested that inhibitory interneurons are the major targets of the mossy fiber system. To study the regulation of mossy fiber-interneuron excitation, we examined unitary and compound excitatory postsynaptic currents in dentate gyrus basket cells, evoked by paired recording between granule and basket cells or extracellular stimulation of mossy fiber collaterals. The application of an associative high-frequency stimulation paradigm induced posttetanic potentiation (PTP) followed by homosynaptic long-term potentiation (LTP). Analysis of numbers of failures, coefficient of variation, and paired-pulse modulation indicated that both PTP and LTP were expressed presynaptically. The Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) did not affect PTP or LTP at a concentration of 10 mM but attenuated LTP at a concentration of 30 mM. Both forskolin, an adenylyl cyclase activator, and phorbolester diacetate, a protein kinase C stimulator, lead to a long-lasting increase in excitatory postsynaptic current amplitude. H-89, a protein kinase A inhibitor, and bisindolylmaleimide, a protein kinase C antagonist, reduced PTP, whereas only bisindolylmaleimide reduced LTP. These results may suggest a differential contribution of protein kinase A and C pathways to mossy fiber-interneuron plasticity. Interneuron PTP and LTP may provide mechanisms to maintain the balance between synaptic excitation of interneurons and that of principal neurons in the dentate gyrus-CA3 network.     

Fast synaptic inhibition promotes synchronized gamma oscillations in hippocampal interneuron networks

Networks of GABAergic interneurons are of critical importance for the generation of gamma frequency oscillations in the brain. To examine the underlying synaptic mechanisms, we made paired recordings from "basket cells" (BCs) in different subfields of hippocampal slices, using transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the parvalbumin promoter. Unitary inhibitory postsynaptic currents (IPSCs) showed large amplitude and fast time course with mean amplitude-weighted decay time constants of 2.5, 1.2, and 1.8 ms in the dentate gyrus, and the cornu ammonis area 3 (CA3) and 1 (CA1), respectively (33-34 degrees C). The decay of unitary IPSCs at BC-BC synapses was significantly faster than that at BC-principal cell synapses, indicating target cell-specific differences in IPSC kinetics. In addition, electrical coupling was found in a subset of BC-BC pairs. To examine whether an interneuron network with fast inhibitory synapses can act as a gamma frequency oscillator, we developed an interneuron network model based on experimentally determined properties. In comparison to previous interneuron network models, our model was able to generate oscillatory activity with higher coherence over a broad range of frequencies (20-110 Hz). In this model, high coherence and flexibility in frequency control emerge from the combination of synaptic properties, network structure, and electrical coupling.     

Frequency-dependent associative long-term potentiation at the hippocampal mossy fiber-CA3 synapse.

The mossy fiber-CA3 synapse displays an N-methyl-D-aspartate-receptor-independent mu-opioid-receptor-dependent form of long-term potentiation (LTP) that is thought not to display cooperativity or associativity with coactive afferents. However, because mossy fiber LTP requires repetitive synaptic activity for its induction, we reevaluated cooperativity and associativity at this synapse by using trains of mossy fiber stimulation. Moderate-, but not low-, intensity trains induced mossy fiber LTP, indicating cooperativity. Low-intensity mossy fiber trains that were normally ineffective in inducing LTP could induce mossy fiber LTP when delivered in conjunction with trains delivered to commissural-CA3 afferents. Associative mossy fiber LTP also could be induced with single mossy fiber pulses when delivered with commissural trains in the presence of a mu-opioid-receptor agonist. Our findings suggest a frequency-dependent variation of Hebbian associative LTP induction that is regulated by the release of endogenous opioid peptides.     

Assessing the role of Ih channels in synaptic transmission and mossy fiber LTP

Hyperpolarization-activated nonselective cation channels (Ih channels) play an important role in the control of membrane excitability and rhythmic neuronal activity. The functional relevance of presynaptic Ih channels in regulating synaptic function, however, is not well established. Recently, it has been proposed [Mellor, J., Nicoll, R. A. & Schmitz, D. (2002) Science 295, 143-147] that presynaptic Ih channels are necessary for hippocampal mossy fiber long-term potentiation (LTP). This observation challenges an alternative model that suggests presynaptic forms of LTP are caused by a direct modification of the transmitter release machinery. Here, we assess the role of Ih in hippocampal mossy fiber LTP as well as cerebellar parallel fiber LTP, forms of potentiation that share common mechanisms. Our results show that after Ih blockade neither mossy fiber LTP nor parallel fiber LTP are affected. Furthermore, Ih does not significantly modify basal excitatory synaptic transmission in the hippocampus, whereas the organic Ih blockers ZD7288 and DK-AH 269 induce a large Ih-independent depression of synaptic transmission. In summary, our results indicate that Ih-mediated persistent changes in presynaptic excitability do not underlie presynaptic forms of LTP.     

Nucleus basalis magnocellularis lesions impair mossy fiber system in rat hippocampus: a quantitative histochemical and ultrastructural study

Lesions of the nucleus basalis magnocellularis (NBM) cause depletion of choline acetyltransferase (ChAT) in the cerebral cortex and behavioral changes consisting of impaired ability to learn avoidance tasks. Since hippocampal mossy fibers (MF) are involved in the elaboration of passive avoidance responses, we analyzed MF by means of Timm's histochemical technique and electron microscopy, to find out whether monolateral lesions of NBM had any effect on MF system. NBM-lesioned rats, 3 weeks after lesioning, showed a significant and progressive decrease in the density of Timm staining as well as significant changes of the morphology of synapic boutons of the MF. These results suggest that, although NBM does not send direct projections to the hippocampus, lesions of this nucleus may have a neurodegenerative effect on the intrahippocampal pathway involved in avoidance responses.