GABAergic and non-GABAergic thalamic, hypothalamic and basal forebrain projections to the ventral oral pontine reticular nucleus: Their implication in REM sleep modulation

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

GABAergic and non-GABAergic thalamic, hypothalamic and basal forebrain projections to the ventral oral pontine reticular nucleus: Their implication in REM sleep modulation Margarita L. Rodrigo-Angulo a,⁎, Susana Herederob , Elisia Rodríguez-Veigab , Fernando Reinoso-Suáreza a

Departamento de Anatomía, Histología y Neurociencia, Facultad de Medicina, UAM, Madrid, Spain Departamento de Anatomía, Facultad de Veterinaria, UCM, Madrid, Spain

b

A R T I C LE I N FO

AB S T R A C T

Article history:

The ventral part of the oral pontine reticular nucleus (vRPO) is a demonstrated site of brainstem

Accepted 26 February 2008

REM-sleep generation and maintenance. The vRPO has reciprocal connections with structures

Available online 10 March 2008

that control other states of the sleep–wakefulness cycle, many situated in the basal forebrain and the diencephalon. Some of these connections utilize the inhibitory neurotransmitter

Keywords:

GABA. The aim of the present work is to map the local origin of the basal forebrain and

Paradoxical sleep

diencephalon projections to the vRPO whether GABAergic or non-GABAergic. A double-

Dorsolateral hypothalamus

labelling technique combining vRPO injections of the neuronal tracer, cholera-toxin (CTB), with

Reticular thalamic nucleus

GAD-immunohistochemistry, was used for this purpose in adult cats. All of the numerous CTB-

Zona incerta

positive neurons in the reticular thalamic and dorsocaudal hypothalamic nuclei were double-

GABA

labelled (CTB/GAD-positive) neurons. Approximately 15%, 14% and 16% of the CTB-positive

Cat

neurons in the zona incerta and the dorsal and lateral hypothalamic areas are, respectively, CTB/GAD-positive neurons. However, only some double-labelled neurons were found in other hypothalamic nuclei with abundant CTB-positive neurons, such as the paraventricular nucleus, perifornical area and H1 Forel field. In addition, CTB-positive neurons were abundant in the central amygdaline nucleus, terminal stria bed nuclei, median preoptic nucleus, medial and lateral preoptic areas, dorsomedial and ventromedial hypothalamic nuclei, posterior hypothalamic area and periventricular thalamic nucleus. The GABAergic and non-GABAergic connections described here may be the morphological pillar through which these prosencephalic structures modulate, either by inhibiting or by exciting, the vRPO REM-sleep inducing neurons during the different sleep–wakefulness cycle states. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

The ventral part of the oral pontine reticular nucleus (vRPO) is a nodal link in the complex neuronal network responsible for

the generation and maintenance of rapid eye movement (REM) sleep (Reinoso-Suárez et al., 1994, 2001) (Fig. 1). The vRPO has reciprocal connections with structures that control the other states of the sleep–wakefulness cycle (Reinoso-Suárez et al.,

⁎ Corresponding author. Departamento de Anatomía, Histología y Neurociencia, Facultad de Medicina, UAM, Arzobispo Morcillo s/n. 28029 Madrid, Spain. Fax: +34 91 3945338. E-mail address: [email protected] (M.L. Rodrigo-Angulo). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.02.095

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Fig. 1 – Location of the tracer injections. Top: schematic drawings of cat brainstem coronal sections at stereotaxic planes AP-2 and AP-3 (Reinoso-Suárez, 1961) show the locations of CTB injections in the vRPO (delineated by a grey line) in cases G1 to G5. The injection of cat G4 (represented in Fig. 2) is delineated with the thicker trace and shows a shaded core. Bottom: microphotograph of the brainstem coronal section of case G1 showing the CTB deposit. AP, antero-posterior plane; BP, brachium pontis; CS, central superior raphe nucleus; DR, dorsal raphe nucleus; IC, inferior colliculus; LcC, locus coeruleus complex; LdT, laterodorsal tegmental nucleus; ML, medial lemniscus; MLF, medial longitudinal bundle; P, pyramidal tract; RPO, oral pontine reticular nucleus; SCP, superior cerebellar peduncle; Tp, tegmentopontine nucleus; vRPO, ventral part of RPO. 1990, 1994, 2001; De la Roza and Reinoso-Suárez, 2000, 2006; De la Roza et al., 2004). These other structures include the basal forebrain–anterior hypothalamic region, the reticular thalamic nucleus and the posterior lateral hypothalamus, which have all long been considered crucial to the physiological phenomena that occur during other states of the sleep– wakefulness cycle. These interconnections utilize a great variety of neurotransmitters and may provide the anatomical basis for alternation of the different states of the sleep– wakefulness cycle in equilibrium of inhibition–excitation (von Economo, 1930; Sterman and Clemente, 1962; Moruzzi, 1972; Reinoso-Suárez and De Andrés, 1976; Steriade et al., 1987; Arcelli et al., 1997, Saper et al., 2001). Microinjections of the GABAA receptor agonist muscimol in the oral pontine reticular nucleus significantly decrease REM

sleep while microinjections of the GABAA receptor antagonist bicuculine increase REM sleep in cats and rats (Xi et al., 1999, 2001, 2004; Manquillo, 2000; Sanford et al., 2003). In vitro experiments in rats have demonstrated that GABA hyperpolarizes vRPO neurons (Núñez et al., 1998), while recent in vivo microdialysis studies have demonstrated the existence of functional GABA transporters in the oral pontine reticular nucleus (Watson et al., 2007). Light (Mugnaini and Oertel, 1985; Ford et al., 1995; De la Roza and Reinoso-Suárez, 2006) and electron microscope studies (De la Roza and Reinoso-Suárez, 2003, 2006; De la Roza et al., 2004) have described GABA-immunoreactive (GABA-ir) neurons and/ or terminals in the vRPO. De la Roza and Reinoso-Suárez (2006) found that 30% of all the vRPO synaptic terminals are GABA-ir, supporting the hypothesis that inhibitory GABAergic terminals

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Fig. 2 – Distribution of CTB-positive and CTB/GAD-positive neurons projecting to vRPO from basal forebrain and diencephalic structures. Drawings schematize coronal sections across basal forebrain and diencephalon from rostral (1) to caudal (6) in case G4 showing the distribution of CTB (small dots) and CTB/GAD (black dots) neurons in basal forebrain, thalamic and hypothalamic structures. Each symbol represents one neuron. AAA, anterior amygdaloid area; AC, anterior commissure; ACe, central amygdaloid nucleus; AHA, anterior hypothalamic area; BNSTl: lateral part of the stria terminalis bed nucleus; BNSTm, medial part of the stria terminalis bed nucleus; Cm: centromedian thalamic nucleus; DcH, dorsocaudal hypothalamic nucleus; DH, dorsal hypothalamic area; DM, dorsomedial thalamic nucleus; DmH, dorsomedial hypothalamic nucleus; Fo, fornix; H1, H1 Forel field; ICa, internal capsule; LaH, lateral habenular nucleus; LG, lateral geniculate nucleus; LGV, ventral lateral geniculate nucleus; LH, lateral hypothalamic area; LPo, lateral preoptic area; MG, medial geniculate nucleus; MN, mammillary nucleus; MnPo, median preoptic nucleus; MPo, medial preoptic area; Mt, mammillothalamic tract; OT, optic tract; PaH, paraventricular hypothalamic nucleus; PeT, periventricular thalamic nucleus; Pf, parafascicular thalamic nucleus; PH, posterior hypothalamic area; PP, pes pedunculi; RB, retroflex bundle; RT, reticular thalamic nucleus; SI, substantia innominata; ST, terminal stria; Tm: tuberomammillary nucleus; VA, ventral anterior thalamic nucleus; VmH, ventromedial hypothalamic nucleus; VP, ventral posterior thalamic nucleus; ZI, zona incerta.

significantly control vRPO neuronal activity. The different densities of these terminals on vRPO neurons suggest the existence of terminals with different origins and physiological

roles. Although some of these GABAergic terminals on vRPO neurons may arise from local neurons, many probably originate from GABA-ir containing structures that are related

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Fig. 3 – Samples of labelled neurons. Microphotographs showing CTB-positive neurons (white arrows) and CTB/GAD-positive double-labelled neurons (black arrows) in reticular thalamic nucleus (A) and dorsal hypothalamic area (B). C: Panoramic view of the dorsocaudal hypothalamic nucleus and detail of the framed area at high magnification of the double-labelled neurons (D). DcH, dorsocaudal hypothalamic nucleus; 3V, third ventricle. Calibration bars: A, 15 μm; B, 20 μm; C, 70 μm; D, 30 μm.

with other states of the sleep–wakefulness cycle, such as the basal forebrain–anterior hypothalamic region, reticular thalamic nucleus, zona incerta or the posterior lateral hypothalamus (Jones, 1975; Oertel et al., 1983; Mugnaini and Oertel, 1985; Yen et al., 1985; Brashear et al., 1986; Gritti et al., 1998, 2003, 2006; Arcelli et al., 1997; Xi et al., 1999; Abrahamson and Moore, 2001; Manns et al., 2001). A GABAergic projection from these structures would support the hypothesis of a specific inhibition of vRPO neurons during other states of the sleep–wakefulness cycle. The aim of the present work is: 1) characterize the projections from basal forebrain, thalamus and hypothalamus to the vRPO, and 2) determine the location of GABA-containing neurons that comprise these projections and that consequently modulate the REM sleep elicited in the vRPO.

2.

Results

The study of CTB deposits revealed that the injection received by each animal (G1–G5) covered all vRPO sectors where microinjections of small amounts (20 nl) and doses (0.04 μg) of a solution of carbachol produce all the bioelectrical and behavioural signs of REM sleep with short latency (Reinoso-Suárez et al., 1990, 1994, 2001; Garzón et al., 1998). All injections were targeted at the AP-2 and AP-3 stereotaxic planes (ReinosoSuárez, 1961) and some of them have slight rostral spreading (Fig. 1). The microscopic study of the double-labelled series showed, in addition to the single GAD-immunoreactive (GAD-ir) neu-

rons, retrogradely labelled CTB-positive and CTB/GAD-positive neurons in the basal forebrain and in the diencephalic structures. In all cases, the distribution of single-labelled GADir neurons did not differ from the classic descriptions in cat and other species (De Biasi et al., 1986; Arcelli et al., 1997; Steriade et al., 1987; Abrahamson and Moore, 2001). These GAD-ir neurons displayed different shapes, sizes, numbers and GADimmunoreaction intensities in the different structures. In the reticular thalamic nucleus they were identified by an elongated shape, the orientation of their dendritic trees in the transverse plane, and their moderate GAD-immunoreaction. The GAD-ir neurons in zona incerta and hypothalamic structures were numerous, round-shaped without well-defined processes, small in size and intensely GAD-reactive; in addition, an intense GAD-reaction also affected many of the processes and terminals occupying the neuropile. The GAD-ir neurons in the thalamic nuclei were also small with intense GAD-reactivity, but they had well-defined processes. A considerable number of CTB-positive neurons were detected in structures or nuclei of the basal forebrain, thalamus and hypothalamus (both ipsi- and contralaterally to the tracer injection) while CTB/GAD-positive neurons were much less abundant and mainly located in the ipsilateral side. These neurons were accurately located using the Nissl stained series and following the Reinoso-Suárez (1961) and the Avendaño and Reinoso-Suárez (1975) atlases to delimit the different structures. In telencephalic structures CTB-positive neurons were located ipsilaterally in the anterior amygdaloid area and central amygdaloid nucleus, the latter structure being entirely

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Table 1 – Numbers and percentages of CTB-positive and CTB/GAD-positive neurons found in ZI, DH, LH and RT G-1 G-2 G-3 G-4 G-5 ZI

DH

LH

RT

Number of neurons CTB 248 272 CTB/GAD 56 38 Total 304 310 GABA Non-GABA CTB 205 176 CTB/GAD 21 28 Total 226 204 GABA Non-GABA CTB 176 212 CTB/GAD 18 62 Total 194 274 GABA Non-GABA CTB CTB/GAD 126 146 Total GABA Non-GABA

275 41 316

233 75 308

281 27 308

X ± SD

%

261.8 ± 20.41 47.4 ± 18.58 309.2 ± 4.38 15.3 84.7

180 19 199

199 54 253

190 42 232

190.0 ± 12.26 32.8 ± 14.8 222.8 ± 21.94 14.7 85.3

158 20 178

153 43 196

133 22 155

166.4 ± 29.73 33.0 ± 19.07 199.4 ± 44.81 16.6 83.4

160

172

145

149.8 ± 17.32 149.8 ± 17.32 100

The results of counting CTB-positive and CTB/GAD-positive neurons in the five cases (G1–G5) are expressed individually and by means of the media ± SE, and by the percentages of GABAergic and non-GABAergic neurons projecting to vRPO from each structure. Data show only the result obtained in the ipsilateral side, since the number of CTB/GAD-positive neurons in the contralateral side was not relevant.

filled (Figs. 2-1 and 2-2). However, the CTB-positive neurons were bilaterally distributed in most basal forebrain structures. The number of CTB -positive neurons in the ipsilateral substantia innominata was quite low but high in the ipsilateral medial and lateral parts of the stria terminalis bed nucleus and medial and lateral preoptic area (Fig. 2-1). The contralateral medial part of the stria terminalis bed nucleus, medial preoptic area and lateral preoptic area showed some CTBpositive neurons while the median preoptic nucleus was filled with the single-labelled CTB-positive neurons (Fig. 2-1). In no case were we able to detect any double-labelled CTB/GADpositive neuron in the above-mentioned basal forebrain structures. In contrast, both CTB-positive and CTB/GAD-positive neurons were detected in diencephalic structures. In the thalamus, the reticular thalamic nucleus displayed a considerable number of CTB/GAD-positive neurons across its antero-posterior extent but only on the ipsilateral side. At rostral levels the neurons of the reticular thalamic nucleus occupied the core of the nucleus while at caudal levels they appeared in its infrageniculate sector, and always intermingled with GADpositive neurons (Figs. 2-2 to 5 and 3-A). Obviously, since no single labelled CTB-positive neuron was identified in this structure, the entire projection from the reticular thalamic nucleus to the vRPO is GABAergic (Table 1). Other thalamic structures showed either only a few CTB-positive neurons, such as the ipsilateral ventral lateral geniculate nucleus, the ipsi- and contralateral lateral habenular nuclei and the ipsilateral parafascicular thalamic nucleus, or else many more

cells, such as the ipsilateral periventricular thalamic nucleus (Fig. 2-6). The ipsilateral zona incerta contains a large number of double-labelled CTB/GAD-positive neurons intermingled with numerous single-labelled neurons, either CTB or GAD-ir, as well as profuse passing fibers. In this case, a few doublelabelled neurons were detected in the contralateral zona incerta (Figs. 2-5 to 6). Only 15.3% of the zona incerta neurons projecting to the vRPO are GABAergic (Table 1). In the hypothalamic structures CTB-positive neurons were very numerous in both the ipsi- and contralateral sides, while double-labelled CTB/GAD-positive neurons were remarkably less abundant, situated in the posterior part of the hypothalamus and had an almost exclusively ipsilateral distribution. The dorsal hypothalamic area, the paraventricular hypothalamic nucleus, the dorsomedial hypothalamic nucleus, the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the posterior hypothalamic area and the perifornical area showed abundant CTB-positive neurons on both sides (Fig. 2-2 to 6); less abundantly, CTB-positive neurons were also found in the anterior hypothalamic area, the H1 Forel field and the tuberommamillary nucleus (Fig. 2). However, the number of CTB/GAD-positive neurons in hypothalamic structures was moderate and practically restricted to the ipsilateral side; these neurons were observed in the dorsal hypothalamic area, most abundantly in its caudal sectors (Figs. 2-4 and 3-C) and constituted 14.7% of its projection to the vRPO (Table 1). In a similar way, 16.6% of the retrogradely labelled neurons distributed in the lateral hypothalamic area were also CTB/ GAD-positive (Table 1). However, only a few double-labelled neurons could be observed in the paraventricular hypothalamic nucleus, H1 Forel field or perifornical area (Fig. 2). At caudal hypothalamic levels close to the third ventricle, a defined small nucleus constituted by small packed cells could be identified by the intense GAD-positive immunoreaction (Fig. 3-B). This nucleus was named the dorsocaudal hypothalamic nucleus in cats by Avendaño and Reinoso-Suárez (1975). A large number of CTB/GAD-positive neurons filled the dorsocaudal hypothalamic nucleus in both the ipsi- and contralateral sides, however, not one single-labelled CTB-positive neuron could be detected in this nucleus (Figs. 2-5 to 6 and 3-B). Consequently, all of the neurons in the dorsocaudal hypothalamic nucleus projecting to vRPO are GABAergic.

3.

Discussion

Numerous studies performed in the last fifty years have demonstrated that the pontine tegmentum plays a critical role in the generation and maintenance of REM sleep through cholinergic mechanisms (Hobson et al., 1975; Baghdoyan et al., 1984, 1987; Vanni-Mercier et al., 1989; Reinoso-Suárez et al., 1990, 1994, 2001; Yamamoto et al., 1990; Jones, 1991; Garzón et al., 1998; Pace-Schott and Hobson, 2002). Experiments in freely moving cats have demonstrated that the vRPO is a well-defined site in the brainstem (Fig. 1) where small-volume microinjections of low doses of the cholinergic agonist carbachol produce enduring periods of REM sleep with short latency; however, the same injections performed dorsal, rostral or caudal to the vRPO pontine structures only evoked

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partial events of REM sleep (Reinoso-Suárez et al., 1990, 1994, 2001; Garzón, 1996; Garzón et al., 1998). Afferent connections to pontine structures situated dorsally or caudally to the vRPO have been identified (Graybiel, 1977; Jones and Yang, 1985; Leichnetz et al., 1989; Fitzpatrick et al., 1989; Reinoso-Suárez et al., 1990, 1994; Garzón, 1996). The patterns of connections that evoked only partial REM sleep events after combined microinjections of a retrograde tracer and carbachol in regions other than vRPO in cats are different from the connection patterns found in animals in which the injections were placed in vRPO and evoked all signs of REM sleep with short latency (Reinoso-Suárez et al., 1990, 1994, 2001; Garzón, 1996). In this study we demonstrate the existence of GABAergic as well as non-GABAergic projections in the pattern of connections to the vRPO from the basal forebrain and diencephalic structures, which are related with sleep–wakefulness cycle control.

3.1.

GABAergic projections to vRPO

De la Roza and Reinoso-Suárez (2006) suggested the hypothesis that inhibitory GABAergic terminals that directly target somata and different sectors of the dendritic tree significantly control vRPO neuron activity. Consequently, GABAergic input could inhibit vRPO REM sleep-inducing neurons during other states of the sleep–wakefulness cycle such as wakefulness or non-REM sleep. Previously De la Roza et al. (2004) demonstrated that about 26% of the projections to the vRPO from the crucial structure for maintaining normal wakefulness, the posterior lateral hypothalamus, are GABAergic. These authors believe that the activation of this hypothalamic GABAergic projection could inhibit the vRPO REM sleep-inducing neurons and thus contribute to the suppression of REM sleep activation during wakefulness. Since a similar suggestion can be made regarding other putative GABAergic projections from sleep related structures in the diencephalon and basal forebrain, an important aim of the present work is focussed in locating the GABAergic neurons in these structures that should be modulating the state of REM sleep elicited by the vRPO in the different phases of the sleep–wakefulness cycle. The main diencephalic source of GABAergic projections to the vRPO is the ipsilateral reticular thalamic nucleus. Tracing studies in the cat have demonstrated projections from the reticular thalamic nucleus and other subthalamic structures, such as the zona incerta, ventral lateral geniculate nucleus and lateral posterior hypothalamus, to the superior colliculus, central grey matter and mesencephalic reticular formation (Grofovà et al., 1978; Edwards et al., 1979; Tortelly and ReinosoSuárez, 1980; Tortelly et al., 1980). Retrograde HRP labelling from the vRPO in the ipsilateral reticular thalamic nucleus has already been reported in cats (Reinoso-Suárez et al., 1990, 1994). The present results confirm the GABAergic nature of these projections from the reticular thalamic nucleus to vRPO. The reticular thalamic nucleus is considered to be responsible for sleep spindle generation, a significant bioelectric component in the non-REM sleep state of the sleep–wakefulness cycle (Steriade et al., 1987; Arcelli et al., 1997; Reinoso-Suárez et al., 2001). Consequently, the inhibitory effect of the reticular

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thalamic nucleus GABAergic projections to the vRPO might contribute to suppressing REM sleep induction during the nonREM sleep state of the sleep–wakefulness cycle. A similar effect may be attributed to the GABAergic projection from the zona incerta to vRPO. The zona incerta is at the crossroads of great ascending and descending tracts, and has the same embryological origin as the reticular thalamic nucleus (Reinoso-Suárez, 1966). It has been demonstrated that ascending inputs from cholinergic brainstem nuclei, which would operate during wakefulness and REM sleep states, suppress the zona incerta GABAergic inhibition of thalamic projection neurons during non-REM sleep (Masri et al., 2006). An equivalent inhibitory mechanism may also be exerted by the zona incerta GABAergic neurons to regulate the vRPO neurons during non-REM sleep. The present work describes for first time a well-defined group of densely packed, small, round and intensely GAD-ir positive neurons in the caudal and dorsal part of the hypothalamus, close to the third ventricle. This group of cells is denominated the dorsocaudal hypothalamic nucleus in the Avendaño and Reinoso-Suárez (1975) stereotaxic cat atlas. Other descriptions of this nucleus have not been found in the revised literature. We have found a consistent bilateral projection to the vRPO that originates from this GABAergic nucleus; remarkably the projection from the dorsocaudal hypothalamic nucleus to the vRPO is entirely GABAergic. The physiological role of this nucleus is still to be elucidated, but it would seem to be related with the hypothalamic waking structures, and, if this is the case, an inhibitory effect by the dorsocaudal hypothalamic nucleus GABAergic projections over the vRPO could contribute to the suppression of REM sleep induction during wakefulness. However, a more likely source of this inhibitory effect would be the CTB/GAD-positive neurons projecting to the vRPO from the dorsal hypothalamic area and lateral hypothalamic area, since these structures have been related to wakefulness maintenance and a large proportion of their neurons are most active during wakefulness (Lin, 2000; Saper et al., 2001, 2005; Sutcliffe and de Lecea, 2002; Koyama et al., 2003). The finding that 14.7% of the dorsal hypothalamic area neurons and 16.6% of the lateral hypothalamic area neurons are CTB/GAD-positive can explain the findings of De la Roza et al. (2004). Their study showed that about 26% of terminals labelled after anterograde tracer injections into the posterolateral hypothalamus affecting both the dorsal hypothalamic and lateral hypothalamic area are GABAergic and form symmetric synapses with vRPO neurons. Activation of descending inhibitory GABAergic influences from these hypothalamic structures would contribute to the suppression of REM sleep triggering during wakefulness. Other hypothalamic nuclei, such as paraventricular hypothalamic nucleus, perifornical area and H1 Forel field, may only a few GABAergic neurons in these nuclei project to vRPO.

3.2.

Non-GABAergic projections to vRPO

We found that the vRPO receives large non-GABAergic projections from the ipsilateral central amygdaline nucleus and from both the medial and lateral parts of the stria terminalis bed nuclei, these being mostly ipsilateral. Although

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a GABAergic population of central amygdaline nucleus neurons projecting to brainstem nuclei related to autonomic functions has been described in the monkey (Jongen-Rêlo and Amaral, 1998), we did not find any reference in the literature regarding the GABAergic nature of these neurons in the cat; in fact, we were not able to detect any GAD-positive neuron in the central amygdaloid nucleus in the present study. NonGABAergic projections from the stria terminalis bed nucleus and central amygdaloid nucleus to brainstem structures related with the organization of REM sleep have been described in the rat (Boissard et al., 2003). Also, the significant projection from the central amygdaloid nucleus to the brainstem and the implication of the amygdala, specifically the central amygdaloid nucleus, in the regulation of REM sleep are well known (Sanford et al., 2006). The central amygdaloid nucleus fires spontaneously during REM sleep; electrical stimulation of this nucleus increases REM sleep while its chemical inhibition decreases REM sleep (Sanford et al., 2002). In conjunction with the present results these earlier descriptions suggest an active influence by the amygdala directly, or through the stria terminalis bed nucleus, on vRPO management of REM sleep. The large non-GABAergic projection from median preoptic nucleus to vRPO reported here is consistent with the description of Gvilia et al. (2006) in rats that increasing REM sleep pressure predominately activates the non-GABAergic neurons in the median preoptic nucleus. This result suggests that these non-GAD-positive neurons of the median preoptic nucleus, which are possibly glutamatergic, exert an excitatory effect directly on vRPO REM sleep generation neurons; a similar effect may be exerted by the scarce non-GAD-positive neurons projecting to the vRPO from the ventral lateral preoptic area and could correspond with the low number of non-GADpositive neurons in the ventral lateral preoptic area activated by REM sleep pressure (Gvilia et al., 2006; Lu et al., 2002). Our hypothesis would support a direct modulation of REM sleep from these preoptic structures rather than the equivalent effect produced by the indirect inhibition of the rostral brainstem aminergic neurons as was proposed by Gvilia et al. (2006). However, the inhibition of this aminergic system through the GAD-positive median preoptic nucleus and ventral lateral preoptic area neurons, as proposed by Gvilia et al. (2006), may be a complementary prerequisite for REM sleep generation. A decrease in the frequency of REM sleep episodes has been described in rats after lesions in the medial preoptic area (Srividya et al., 2006); we have found abundant projections to the vRPO from this area. This connection may be the anatomical substrate through which the medial preoptic area contributes to the maintenance of REM sleep episode frequency. The vRPO receives very important non-GABAergic afferent connections from the zona incerta, dorsal hypothalamic area, lateral hypothalamic area, dorsomedial hypothalamic nucleus, ventromedial hypothalamic nucleus, paraventricular hypothalamic nucleus, posterior hypothalamic area, tuberomammilary nucleus, periventricular thalamic nucleus and perifornical area; this mostly ipsilateral but also bilateral connection confirms previous reports of diencephalic afferents to the vRPO in the cat (Reinoso-Suárez et al., 1994) and rat (Núñez et al., 2006). Histamine, galanin, glutamate, melaninconcentrating hormone and orexin-positive neurons have all

been described, in addition to GABAergic neurons, in these hypothalamic nuclei in the cat and/or rat (Mugnaini and Oertel, 1985; Airaksinen et al., 1992; Lin, 2000; Elias et al., 1998; Sutcliffe and de Lecea, 2002). Probably, most projections from these neurons form asymmetric synapses on the vRPO neurons (De la Roza et al., 2004) and some might originate in the many hypothalamic neurons that are active during REM sleep (Koyama et al., 2003): for example, the distribution of the rat hypothalamic neurons that express Fos during the rebound of REM sleep following REM sleep deprivation, is similar in the zona incerta and hypothalamus to that of the CTB-positive neurons reported here in the cat (Verret et al., 2003). The vRPO terminals of these diencephalic neurons would excite the vRPO neurons that generate REM sleep. On the other hand, De la Roza et al. (2004) showed that a large number of the nonGABAergic hypothalamic terminals form symmetric synapses on the vRPO neurons. These terminals may originate in the hypothalamic neurons that are most active during either wakefulness or non-REM sleep (Koyama et al., 2003) and utilize one of the many hypothalamic neurotransmitters other than GABA to inhibit vRPO neurons during those states of the sleep–wakefulness cycle. Interestingly, all or part of the prosencephalic connections described here may be the anatomical substrate necessary for forebrain homeostasis of REM sleep that was proposed by De Andrés et al. (2003).

Fig. 4 – Summary of the basal forebrain and diencephalic connections to the vRPO. In the scheme, open circles represent non-GABAergic neurons and black circles GABAergic neurons. The grey circle represents the serotonergic neurons. Dashes and arrows respectively represent symmetric (inhibitory) and asymmetric (excitatory) synapses. Reticular thalamic and zona incerta GABAergic neurons may inhibit vRPO REM sleep-inducing neurons during non-REM sleep while hypothalamic GABAergic neurons would be active during wakefulness. A large number of the basal forebrain and hypothalamic structures exert a direct excitatory effect on the organization of REM sleep through the asymmetric synapses on the vRPO neurons. On the other hand, hypothalamic, zona incerta and, basal forebrain non-GABAergic neurons, represented in the bottom of the figure, could inhibit vRPO REM sleep-inducing neurons, either directly by forming symmetric synapses or indirectly through the excitation of local GABAergic and/or serotonergic vRPO neurons. RT, reticular thalamic nucleus; ZI, zona incerta.

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3.3.

Concluding remarks

Taken as a whole, our data demonstrate extensive connections from the basal forebrain, hypothalamus, zona incerta and reticular thalamic nucleus to vRPO. Only projections from the reticualr thalamic nucleus, zona incerta and lateral– posterior hypothalamus are GABAergic and may possibly inhibit the vRPO neurons that generate and maintain REM sleep. The reticular thalamic nucleus and zona incerta GABAergic neurons may be related with the organization of non-REM sleep while lateral–posterior hypothalamic neurons would be related with wakefulness (Fig. 4). The abundant projections from the basal forebrain and anterior hypothalamus as well as a good part of the projections from the posterior lateral hypothalamus and zona incerta to the vRPO are non-GABAergic (Fig. 4). According to previous results in rats and cats, many of these non-GABAergic projections would excite vRPO neurons through asymmetric synapses, thereby modulating REM sleep generation and homeostasis. However, many of these non-GABAergic projections would inhibit REM sleep-inducing vRPO neurons during the other states of the sleep–wakefulness cycle. This inhibition could be exerted either directly through non-GABAergic terminals forming symmetric synapses on the vRPO neurons (De la Roza et al., 2004), or indirectly through the activation of local serotonergic (Rodrigo-Angulo et al., 2000) and/or GABAergic vRPO neurons in a mechanism that would be similar to the one proposed for the hypocretinergic neurons of the lateral– posterior hypothalamus in the rat (Núñez et al., 2006) (Fig. 4).

4.

Experimental procedures

Following the European Community Council Directive of 24 November 1986 (86/609/EEC) and with the approval of the “Comité para Cuidado y Uso de Animales de Experimentación (Universidad Autónoma de Madrid)”, experiments were carried out in five adult male cats weighing 3.5–4 kg obtained from authorized specialized farms. Efforts were made to minimize animal suffering as well as to reduce the number of animals used. At least five cats are needed to ensure that the tracer injections will have covered a good part of the vRPO. The GABAergic afferent connections from the basal forebrain and diencephalic structures to the vRPO were detected using a double-labelling technique combining injections of the neuronal retrograde tracer cholera toxin B Subunit Gold Conjugate (CTB) (List Biological Laboratories, Campbell, CA) with glutamic acid decarboxylase (GAD) immunohistochemistry, the latter being a reliable marker of GABAergic neurons. Animals were anesthetized with 0.1 cm3/kg of a mixture of intraparenteral ketamine and medetomidine, plus 0.1 cm3/kg of subcutaneous atropine. Aimed at the stereotaxic coordinates of the vRPO (Reinoso-Suárez, 1961) (Fig. 1), each animal received, by means of a 1 μl Hamilton syringe, 1 μl of the tracer (at 50% concentration in bi-distillated water), which is the amount necessary to obtain reliable transport from brainstem to prosencephalon in the cat. At the end of the surgery anesthesia was reversed with 0.05 cm3/kg of intraperitoneal atipamezol and animals were housed in appropriate cages receiving antibiotics and analge-

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sics when necessary. After 10 days survival time animals were deeply anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.3 followed by increasing concentrations of sucrose solutions in the same buffer. Once removed, brains were separated at antero-posterior stereotaxic plane +6 into an anterior block containing the forebrain and a posterior block containing the brainstem, and stored in a 30% sucrose solution for one week for cryopreservation. Anterior and posterior blocks were sectioned at 40 μm on a cryostat and the sections collected in four or three consecutive series, respectively. Series from the posterior block were processed for CTB development and Nissl staining, leaving a third series as a reserve. Anterior block series were processed for CTB development, double labelling and Nissl staining. Following a modification of the Llewellyn-Smith et al. (1990) method, series to be processed for CTB were dehydrated in 50% ethanol for 20 min, washed for 1 h in citrate–acetate buffer at pH 5.5, developed with an IntenSE BL silver enhancement kit (Amersham Biosciences, Buckinghamshire, UK) at three intervals of 20 min each with intermediate rinses, and fixed in 5% sodium tiosulphate. Series to be double-labelled were first processed for CTB and, after storage overnight in phosphate buffer, were incubated with 1:100 mouse anti-GAD antiserum (GAD-6 64 KDa subunit monoclonal antibody, developed by Dr. Gottlieb, DSHB, Iowa University, a stain that has been proven to be reliable for visualizing GABAergic neurons in brain; i.e. www.uiowa.edu/ dshbwww/gad6.html for further information) in a solution containing 5% bovine serum albumin and 30% normal horse serum in saline phosphate buffer for 72 h. Then, sections were incubated in 1:200 biotinylated horse anti-mouse (Chemicon, Temecula, CA) for 2 h and in Elite ABC kit (Vector Laboratories Inc., Burlingame, CA) for 1.5 h before development with 0.05% 3-3′ DAB and 0.003% H2O2. Nissl-stained series were used to delimit structures following the Reinoso-Suárez (1961), and Avendaño and ReinosoSuárez (1975) atlases. The CTB and double-labelled sections were studied under dark and/or bright field illumination at 40x using a Zeiss Axioskop microscope. Images in Fig. 3 were taken through a Spot Insight Color camera attached to the microscope. No further filtering or color/contrast/brightness enhancement was applied to any of the images. Canvas 10.3.9 (Deneba software, FL) was used to crop and align the images in the plate. Quantification of the data was obtained by counting both neuronal profiles, CTB-positive neurons and CTB/GAD-positive neurons, found in reticular thalamic nucleus, zona incerta, and, dorsal and lateral hypothalamic areas, in all sections of the series processed for double-labelling in each animal.

Acknowledgments The GAD-6 monoclonal antibody developed by DR. D.I. Gottlieb was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The authors thank C. Warren for revising English language use. The technical assistance of M. Callejo, G. de la Fuente and R. Sánchez-Lozano

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is gratefully acknowledged. Supported by Spanish Grant BFU 2006-07430.

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