Francisco E. Olucha-Bordonau

Cytoarchitecture and efferent projections of the nucleus incertus of the rat.

The nucleus incertus is located caudal to the dorsal raphe and medial to the dorsal tegmentum. It is composed of a pars compacta and a pars dissipata and contains acetylcholinesterase, glutamic acid decarboxylase, and cholecystokinin‐positive somata. In the present study, anterograde tracer injections in the nucleus incertus resulted in terminal‐like labeling in the perirhinal cortex and the dorsal endopyriform nucleus, the hippocampus, the medial septum diagonal band complex, lateral and triangular septum medial amygdala, the intralaminar thalamic nuclei, and the lateral habenula. The hypothalamus contained dense plexuses of fibers in the medial forebrain bundle that spread in nearly all nuclei. Labeling in the suprachiasmatic nucleus filled specifically the ventral half. In the midbrain, labeled fibers were observed in the interpeduncular nuclei, ventral tegmental area, periaqueductal gray, superior colliculus, pericentral inferior colliculus, pretectal area, the raphe nuclei, and the nucleus reticularis pontis oralis. Retrograde tracer injections were made in areas reached by anterogradely labeled fibers including the medial prefrontal cortex, hippocampus, amygdala, habenula, nucleus reuniens, superior colliculus, periaqueductal gray, and interpeduncular nuclei. All these injections gave rise to retrograde labeling in the nucleus incertus but not in the dorsal tegmental nucleus. These data led us to conclude that there is a system of ascending projections arising from the nucleus incertus to the median raphe, mammillary complex, hypothalamus, lateral habenula, nucleus reuniens, amygdala, entorhinal cortex, medial septum, and hippocampus. Many of the targets of the nucleus incertus were involved in arousal mechanisms including the synchronization and desynchronization of the theta rhythm. J. Comp. Neurol. 464:62–97, 2003.

Nucleus incertus contribution to hippocampal theta rhythm generation

The hippocampal theta rhythm is generated by the pacemaker activity of the medial septum‐diagonal band of Broca (MS/DBB) neurons. These nuclei are influenced by brainstem structures that modulate the theta rhythm. The aim of the present work is to determine whether the nucleus incertus (NI), which has important anatomical connections with the MS/DBB, contributes to the hippocampal theta rhythm generation in rats. Hippocampal field activity was recorded in urethane‐anaesthetized rats. Electrical stimulation of the NI not only evoked theta rhythm in the hippocampus, but also decreased the amplitude of delta waves. Unit recordings in the NI revealed either a non‐rhythm discharge pattern in most neurons (76%), or a rhythm activity at 13–25 Hz in the remaining neurons. The firing rate of these neurons increased during the presence of theta rhythm evoked by either sensory or reticularis pontis oralis nucleus (RPO) stimulation. Electrolytic lesions of NI, or the microinjection of the γ‐aminobutyric acid (GABA)A agonist muscimol, abolished the theta rhythm evoked by RPO stimulation. Consequently, the NI may be a relay station between brainstem structures and the MS/DBB in the control of the hippocampal theta rhythm generation.

LRRK2 is expressed in areas affected by Parkinson's disease in the adult mouse brain

The leucine‐rich repeat kinase 2 (LRRK2) gene was recently found to have multiple mutations that are causative for autosomal dominant inherited Parkinsons disease (PD). Previously, we used Northern blot analysis to show that this gene was expressed in the cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe and caudate putamen. However, a more comprehensive map of LRRK2 mRNA localization in the central nervous system is still lacking. In this study we have mapped the distribution of the mRNA encoding for LRRK2 using nonradioactive in situ hybridization. We detected a moderate expression of this PD‐related gene throughout the adult B2B6 mouse brain. A stronger hybridization signal was observed in deep cerebral cortex layers, superficial cingulate cortex layers, the piriform cortex, hippocampal formation, caudate putamen, substantia nigra, the basolateral and basomedial anterior amygdala nuclei, reticular thalamic nucleus and also in the cerebellar granular cell layer. Given that LRRK2 mRNA is highly enriched in motor systems and also is expressed in other systems, we may conclude that mutations in LRRK2 may affect several motor and nonmotor structures that may play an important role in the development of PD.

Heterogeneous responses of nucleus incertus neurons to corticotrophin‐releasing factor and coherent activity with hippocampal theta rhythm in the rat

•  The nucleus incertus (NI) is a stress and arousal responsive, hindbrain region involved in ascending control of septohippocampal theta rhythm. •  NI neurons express high levels of the neuropeptide relaxin‐3 and corticotrophin‐releasing factor (CRF) receptor‐1 (CRF‐R1). •  We report the first in‐depth characterization of NI neurons, using in vivo and in vitro electrophysiological techniques, which reveal a population of relaxin‐3‐containing NI neurons activated by CRF via postsynaptic CRF‐R1 and a non‐relaxin‐3 neuron population inhibited or unaffected by CRF. •  Relaxin‐3 NI neurons exhibit strong phase‐locked firing with the ascending phase of hippocampal theta oscillations. •  These findings suggest the NI is a heterogeneous neuronal population and key site of CRF action with the capacity to modulate cognition in response to stress.

Distribution and targets of the relaxin-3 innervation of the septal area in the rat

Neural tracing studies have revealed that the rat medial and lateral septum are targeted by ascending projections from the nucleus incertus, a population of tegmental GABA neurons. These neurons express the relaxin‐family peptide, relaxin‐3, and pharmacological modulation of relaxin‐3 receptors in medial septum alters hippocampal theta rhythm and spatial memory. In an effort to better understand the basis of these interactions, we have characterized the distribution of relaxin‐3 fibers/terminals in relation to different septal neuron populations identified using established protein markers. Dense relaxin‐3 fiber plexuses were observed in regions of medial septum containing hippocampal‐projecting choline acetyltransferase (ChAT)‐, neuronal nitric oxide synthase (nNOS)‐, and parvalbumin (PV)‐positive neurons. In lateral septum (LS), relaxin‐3 fibers were concentrated in the ventrolateral nucleus of rostral LS and the ventral nucleus of caudal LS, with sparse labeling in the dorsolateral and medial nuclei of rostral LS, dorsal nucleus of caudal LS, and ventral portion nuclei. Relaxin‐3 fibers were also observed in the septofimbrial and triangular septal nuclei. In the medial septum, we observed relaxin‐3‐immunoreactive contacts with ChAT‐, PV‐, and glutamate decarboxylase‐67‐positive neurons that projected to hippocampus, and contacts between relaxin‐3 terminals and calbindin‐ and calretinin‐positive neurons. Relaxin‐3 colocalized with synaptophysin in nerve terminals in all septal areas, and ultrastructural analysis revealed these terminals were symmetrical and contacted spines, somata, dendritic shafts, and occasionally other axonal terminals. These data predict that this GABA/peptidergic projection modulates septohippocampal activity and hippocampal theta rhythm related to exploratory navigation, defensive and ingestive behaviors, and responses to neurogenic stressors. J. Comp. Neurol. 520:1903–1939, 2012.

Regional distribution of the leucine-rich glioma inactivated (LGI) gene family transcripts in the adult mouse brain.

The leucine-rich glioma inactivated (LGI) gene subfamily contains four highly conserved members (LGI1, 2, 3 and 4), which have been described in human, mouse and other mammalians. Although their main roles remain unknown, LGI1 gene mutations have been found in human partial temporal lobe epilepsy. Moreover, previous studies showed that the products of these genes exert their function in the nervous system. The anatomical distribution of these gene transcripts in the brain might give some insight to elucidate their possible function. In this study, the pattern of expression of the four LGI genes was assessed in the brain of C57BL/6J adult mice by in situ hybridization. We found that the LGI1 transcript is mainly expressed in the dentate gyrus and CA3 field of the hippocampus. LGI2 and LGI4 genes, which showed a similar pattern of distribution with minor differences, were mostly expressed in the medial septal area, thalamic reticular nucleus and substantia nigra pars compacta. LGI3-expressing cells were distributed widespread, but were more consistently observed in the hippocampal formation, thalamic and hypothalamic nuclei, substantia nigra and reticular formation. In summary, LGI1 gene expression is very restricted to intrahippocampal circuitry, which might be related to its involvement in temporal lobe epilepsy. The patterns of expression of LGI2 and LGI4 genes are very similar and their distribution in the vertical limb of the diagonal band and in putative hippocampal interneurons suggests that the function of these genes might be related to the generation of hippocampal theta rhythm. Finally, LGI3 gene widespread expression in the brain suggests that its transcripts might be involved in a common cellular process present in different neuronal types.

Anatomical evidence for a ponto-septal pathway via the nucleus incertus in the rat

Hippocampal theta activity is involved in sensory-motor integration and constitutes a functional basis for mnemonic functions. The medial septum-diagonal band of Broca (MS/DBv) is a key structure as pacemaker of the oscillation. In addition, some brainstem reticular structures are crucial for the activation of MS/DBv. Specifically, the nucleus reticularis pontis oralis (RPO) is considered the most effective pontine site for eliciting theta rhythm. Nevertheless, its connection with the MS/DBv is not direct. A previous study by our group pointed out that the nucleus incertus (NI) could be considered as a relay in this multisynaptic pathway. From this study, the stimulation of RPO increased the discharge rate of NI neurons in anesthetized rats and the lesion of the NI suppressed the RPO-elicited hippocampal theta. Those findings suggested a projection from RPO to NI, although the existing literature did not support this hypothesis. In order to clarify the dichotomy between the anatomical and the electrophysiological data, we performed a set of tracing studies. Anterograde tracer injections into RPO showed a profuse projection to NI. This connection was confirmed by retrograde tracer injections into NI. Injections of retrograde tracer in MS/DBv confirmed the intense NI-MS/DBv projection. Furthermore, simultaneous injections of anterograde and retrograde tracers into RPO and MS/DBv respectively resulted in a high-correlated pattern of terminal-like fibers over labeled somata in the NI. This study provides the first anatomical evidence of a ponto-septal pathway via the NI that contributes to generation and modulation the hippocampal theta activity.

The chemical architecture of the rat's periaqueductal gray based on acetylcholinesterase histochemistry: a quantitative and qualitative study.

The chemoarchitecture of the periaqueductal gray has been extensively studied, based on acetylcholinesterase reaction and comparing it to other chemical markers. We have divided the periaqueductal gray into four main longitudinal columns, namely dorsomedial, dorsolateral, lateral and ventrolateral. We also identified the dorsal midline column, the supraoculomotor cap and the juxta-aqueductal ring. The acetylcholinesterase gave rise to a strong reaction in the outer half of the lateral column, the outer half of the dorsomedial column, the supraoculomotor cap and the ventral half of the juxta-aqueductal ring. This labeling was in part complementary to that of the NADPH diaphorase and allowed the lateral column to be differentiated from the ventrolateral column. However, the inner half of both lateral and ventrolateral columns displayed the same chemical properties including acetylcholinesterase, tyrosine hydroxilase and serotonin. Thus, from the chemical view, these inner halves should be considered as one different region. Finally, the juxta-aqueductal ring was composed of two clearly different halves, i.e. dorsal and ventral. The dorsal half did not show any clear differences from the above columns and was negative for acetylcholinesterase, NADPH diaphorase and tyrosine hydroxilase, while the ventral half was clearly different from the lateral and ventrolateral columns and displayed a positive reaction to all those chemical markers. From these results, we strongly suggest the use of acetylcholinesterase histochemistry as a tool for accurate parcellation of the periaqueductal gray.

The effect of long context exposure on cued conditioning and c-fos expression in the rat forebrain

The c-fos expression was used to study the neural substrates of the cued fear conditioning acquisition, preceded by a short exposure versus a long exposure to the conditioning context. A long-context exposure (either during the night or during the day) prior to conditioning, was associated with low freezing in the learning test. Differences in the c-fos expression of CA1, CA3, BL Amygdala, LS and BNST were found between the short- or long-context groups with a pre-exposure before cued conditioning. Ce Amygdala showed no differences in the c-fos expression labeling. We reported the hippocampal c-fos activation during the cued fear conditioning acquisition. Specifically, the CA1 activation could be related with the context-US processing during the CS-US association acquisition, which might prove that the CS-US associations cannot be made without an integrated context participating. The results showed that a long-context exposure prior to cued conditioning produces an inhibition of the CR (freezing), and this phenomenon is related with a specific c-fos expression in CA1, CA3, BL Amygdala, LS and BNST during the fear acquisition.

Chemical divisions in the medial geniculate body and surrounding paralaminar nuclei of the rat: quantitative comparison of cell density, NADPH diaphorase, acetyl cholin esterase and basal expression of c-fos

Quantitative methods of cell density, the intensities of both acetyl cholinesterase (AChE) and NADPH diaphorase (NADPHd), as well as the basal expression of c-fos, have been carried out in order to study the anatomical divisions of the medial geniculate body (MGB) and the group of nuclei located ventromedially to the MGB called the paralaminar complex (PL). The MGB was composed of the dorsal (MGd), and the ventral (MGv) divisions. We included the medial, or the magnocellular division (MGm), in the PL complex. MGd was composed of a dorsolateral (DL) core and a belt. The belt was composed of the suprageniculate (SG), the deep dorsal (DD), the caudo-medial (CM) and the caudo-dorsal (CD) nuclei. In the MGv, the basal expression of c-fos was the only way to trace a clear boundary between the ovoid (Ov) and the ventrolateral (VL) divisions. However, the marginal zone (MZ) was clearly and contrastingly different. The PL was considered to be composed of: the MGm, the posterior intralaminar nucleus (PIN), the peripeduncular nucleus (PP) and the nucleus subparafascicularis lateralis (SPFL). The MGm and the PIN share most of the chemical features, meanwhile both SPFL and PP displayed different patterns of NADPHd reactivity. The study of cell density on Giemsa stained sections confirmed main divisions of the area. AChE and NADPHd methods allowed the main MGB divisions to be discriminated. The differences between subdivisions were emphasized when cell density and c-fos activity were quantified in each nucleus. Each MGB division displayed a different pattern of c-fos activity under basal conditions. Thus, c-fos basal expression was a particular feature in each MGB or PL nucleus.