Amparo Ruiz-Torner

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.

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.

Regular theta-firing neurons in the nucleus incertus during sustained hippocampal activation

This paper describes the existence of theta‐coupled neuronal activity in the nucleus incertus (NI). Theta rhythm is relevant for cognitive processes such as spatial navigation and memory processing, and can be recorded in a number of structures related to the hippocampal activation including the NI. Strong evidence supports the role of this tegmental nucleus in neural circuits integrating behavioural activation with the hippocampal theta rhythm. Theta oscillations have been recorded in the local field potential of the NI, highly coupled to the hippocampal waves, although no rhythmical activity has been reported in neurons of this nucleus. The present work analyses the neuronal activity in the NI in conditions leading to sustained hippocampal theta in the urethane‐anaesthetised rat, in order to test whether such activation elicits a differential firing pattern. Wavelet analysis has been used to better define the neuronal activity already described in the nucleus, i.e., non‐rhythmical neurons firing at theta frequency (type I neurons) and fast‐firing rhythmical neurons (type II). However, the most remarkable finding was that sustained stimulation activated regular‐theta neurons (type III), which were almost silent in baseline conditions and have not previously been reported. Thus, we describe the electrophysiological properties of type III neurons, focusing on their coupling to the hippocampal theta. Their spike rate, regularity and phase locking to the oscillations increased at the beginning of the stimulation, suggesting a role in the activation or reset of the oscillation. Further research is needed to address the specific contribution of these neurons to the entire circuit.

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.

Distribution of basal-expressed c-fos-like immunoreactive cells of the periaqueductal grey matter of the rat.

The distribution of c-fos proto-oncogene expression has been studied in the periaqueductal grey matter (PAG) of non-intentional-stimulated rats by immunohistochemistry. A mean number of 53 +/- 9 Fos-like immunoreactive cells per hemiPAG, distributed into three groups, dorsolateral, lateral and ventrolateral, was found. The dorsolateral and the lateral groups appeared along the entire rostrocaudal PAG, whereas the ventrolateral group appeared only in the caudal half. These results reveal new data on the number and extent of the longitudinal columns within the hypothetical organization of the PAG. They also indicate a continuous activation of a significant population of neurones in the PAG, in agreement with the suggested role of the PAG in the modulation of internal information.

Morphologic variations in the pineal gland of the albino rat after a chronic alcoholisation process

We studied the effect of alcohol on the pineal gland of 48 male Wistar rats. Animals were divided into control and experimental groups. The experimental group underwent a previous progressive alcoholisation period with ethanol diluted in water at a concentration of 40%. Animals were sacrificed at 3, 6, 9 and 12 months, and the ultrastructure, karyometric indices, and number of synaptic bodies in the pineal gland were analysed. The results showed progressive morphologic alterations in the ethanol-treated animals, which culminated in fatty degeneration of the pineal parenchyma after 6 months. The karyometric indices decreased in both the central and peripheral areas compared with the control group. Moreover, the seasonal rhythmicity observed in the controls disappeared in the experimental groups, whose number of different populations of synaptic bodies (synaptic ribbons and synaptic spherules) considerably lowered with inversion of their normal seasonal rhythm. These results support that chronic alcoholisation leads to fatty degeneration of the pineal parenchyma, and a considerable alteration in nuclear functional rhythms and synaptic bodies.