Alan G. Watts

Basic organization of projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis in adult rat brain

The organization of axonal projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis (BST) was characterized with the Phaseolus vulgaris‐leucoagglutinin (PHAL) anterograde tracing method in adult male rats. Within the BST, the oval nucleus (BSTov) projects very densely to the fusiform nucleus (BSTfu) and also innervates the caudal anterolateral area, anterodorsal area, rhomboid nucleus, and subcommissural zone. Outside the BST, its heaviest inputs are to the caudal substantia innominata and adjacent central amygdalar nucleus, retrorubral area, and lateral parabrachial nucleus. It generates moderate inputs to the caudal nucleus accumbens, parasubthalamic nucleus, and medial and ventrolateral divisions of the periaqueductal gray, and it sends a light input to the anterior parvicellular part of the hypothalamic paraventricular nucleus and nucleus of the solitary tract. The BSTfu displays a much more complex projection pattern. Within the BST, it densely innervates the anterodorsal area, dorsomedial nucleus, and caudal anterolateral area, and it moderately innervates the BSTov, subcommissural zone, and rhomboid nucleus. Outside the BST, the BSTfu provides dense inputs to the nucleus accumbens, caudal substantia innominata and central amygdalar nucleus, thalamic paraventricular nucleus, hypothalamic paraventricular and periventricular nuclei, hypothalamic dorsomedial nucleus, perifornical lateral hypothalamic area, and lateral tegmental nucleus. Moderately dense inputs are found in the parastrial, tuberal, dorsal raphé, and parabrachial nuclei and in the retrorubral area, ventrolateral division of the periaqueductal gray, and pontine central gray. Light projections end in the olfactory tubercle, lateral septal nucleus, posterior basolateral amygdalar nucleus, supramammillary nucleus, and nucleus of the solitary tract. These and other results suggest that the BSTov and BSTfu are basal telencephalic parts of a circuit that coordinates autonomic, neuroendocrine, and ingestive behavioral responses during stress. J. Comp. Neurol. 436:430–455, 2001.

Comparison of melanin-concentrating hormone and hypocretin/orexin mRNA expression patterns in a new parceling scheme of the lateral hypothalamic zone

A high-resolution spatial distribution analysis of hypothalamic neurons expressing melanin-concentrating hormone or hypocretin/orexin was performed in adult male rats with in situ hybridization cytochemistry. For the analysis, a new parcellation of the lateral zone with some two-dozen regions was used, and distributions were plotted on 15 transverse reference levels through the hypothalamus. Qualitatively the results confirm earlier, much lower resolution mapping studies, although some discrepancies are clarified. Previous work indicates that each of these cell populations is far from homogeneous, and the present results should help establish a framework for clarifying more precisely how they are differentiated and organized in terms of axonal input-output relationships and gene expression patterns, and for defining precise relationships with other hypothalamic neuron populations.

The Impact of Physiological Stimuli on the Expression of Corticotropin-Releasing Hormone (CRH) and Other Neuropeptide Genes ☆ ☆☆

The article reviews some of the recent work showing how physiological stimuli act to alter neuropeptide gene expression. It describes how neural and humoral factors activated by physiological stimuli interact with the mechanisms regulating neuropeptide gene expression in neurons with either vascular (neurosecretory) or cellular (centrally directed) synapses. Although the focus will be on corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus, comparisons will be made between this neurosecretory cell group and others that express this gene. The regulation of neuropeptide genes colocalized in neurons that synthesize CRH is also considered. The review begins with a brief historical introduction, placing peptides in the overall functional perspective of neurosecretory and centrally directed neurons. It then describes studies using in vitro preparations that reveal details of the signal transduction mechanisms responsible for altering the expression of neuropeptide genes. For the CRH gene they are providing the foundations for future work on how physiological stimuli alter mRNA levels in the whole animal. Physiological stimuli provide a very broad range of signals to neuropeptide neurons commensurate with the wide variety of motor responses they initiate. One important humoral signal impacting neuropeptide neurons is plasma corticosterone, and many workers have addressed this aspect of its function. Corticosterone appears capable of interacting with at least two different neuronal mechanisms to regulate CRH mRNA levels: one is clearly seen in paraventricular neurosecretory neurons, where increasing plasma corticosteroid reduces CRH mRNA levels; the other, seen in neurons in the central nucleus of the amygdala, acts to increase them. Since physiological stimuli present a complex mixture of humoral and neural signals to the CNS, integration of these two signal types is a critical aspect of peptide metabolism that requires detailed attention. Studies that are beginning to address this important question are described. Circadian influences play an important role in organizing homeostatic processes, and their influence on CRH gene expression is considered. The viscerosensory-motor integration associated with dehydration offers a useful model for investigating the role of peptides in neuronal function and motor architecture. Much of our work has concentrated on how peptide genes are regulated by alterations to fluid homeostasis, and these studies, along with those of other investigators, are described in this integrative context. Finally, consideration is given to the many studies that have addressed the impact of nonviscerosensory stimulation on neuropeptide gene expression.

Sweet talk in the brain: Glucosensing, neural networks, and hypoglycemic counterregulation

Glucose is the primary fuel for the vast majority of cells, and animals have evolved essential cellular, autonomic, endocrine, and behavioral measures to counteract both hypo- and hyperglycemia. A central component of these counterregulatory mechanisms is the ability of specific sensory elements to detect changes in blood glucose and then use that information to produce appropriate counterregulatory responses. Here we focus on the organization of the neural systems that are engaged by glucosensing mechanisms when blood glucose concentrations fall to levels that pose a physiological threat. We employ a classic sensory-motor integrative schema to describe the peripheral, hindbrain, and hypothalamic components that make up counterregulatory mechanisms in the brain. We propose that models previously developed to describe how the forebrain modulates autonomic reflex loops in the hindbrain offer a reasoned framework for explaining how counterregulatory neural mechanisms in the hypothalamus and hindbrain are structured.

Understanding the neural control of ingestive behaviors : Helping to separate cause from effect with dehydration-associated anorexia

Eating and drinking are motivated behaviors that are made up of coordinated sets of neuroendocrine, autonomic, and behavioral motor events. Although the spinal cord, hindbrain, and hypothalamus contain the motor neurons and circuitry sufficient to maintain the reflex parts of these motor events, inputs from the telencephalon are required to furnish the behavioral components with a motivated (goal-directed) character. Each of these motor events derives from the complex interaction of a variety of sensory inputs with groups of neural networks whose components are distributed throughout the brain and collectively support motor expression and coordination. At a first approximation based on a variety of data, these networks can be divided into three groups: networks that stimulate, those that inhibit, and those that disinhibit motor functions. A fourth contributor is the circadian timing signal that originates in the hypothalamic suprachiasmatic nucleus and provides the temporal anchor for the expression of all behaviors. This article discusses the nature of these networks using neuroanatomical (tract-tracing and neuropeptide in situ hybridization), endocrine, and behavioral evidence from a variety of experimental models. A persistent problem when studying the control of food intake from a neural systems perspective has been the difficulty in separating those neuronal changes that result in hunger from those that are as a consequence of eating. To address this problem, dehydration-associated anorexia is presented as a particularly useful experimental model because it can be used to distinguish between neural mechanisms underlying anorexia and those changes that occur as a consequence of anorexia. The article concludes by highlighting the potential role of neuropeptidergic action in the operation of these networks, using forebrain neuropeptidergic innervation of the parabrachial nucleus as an example.

Dehydration-associated anorexia : Development and rapid reversal

Dehydration in rats results in anorexia that is proportional to the degree of dehydration. The aims of this study were first, to determine when anorexia develops in response to drinking hypertonic (2.5%) saline for 4 days; and second, to determine the organization of ingestive behaviors after access to water is resumed. Body weights, food, and fluid intake were measured morning and evening before, during, and after a 4-day period of dehydration caused by drinking hypertonic saline. A profile of the behaviors expressed immediately after rehydration was determined. The data make three points. First, dehydration-associated anorexia does not emerge until the second night of dehydration when the composition of the fluid compartments can no longer be homeostatically buffered. Second, dehydration reduces the amount food eaten nocturnally, but leaves diurnal food consumption largely unaffected. Animals very rapidly return to predehydration nocturnal ingestion patterns, whereas the amounts of food and water ingested during the day are significantly increased. Increased diurnal food intake may play a significant role in normalizing metabolism after dehydration. Finally, anorexia is reversed within minutes of rehydration. The data suggest a model where dehydration simultaneously activates two sets of circuits within the brain that will independently stimulate or inhibit feeding. Eating is inhibited during dehydration through the action of a set of inhibitory circuits, which masks the output of circuits that stimulate eating. However, when drinking water resumes, sensory inputs to these circuits rapidly release the inhibition and allow eating to proceed freely.

Gonadal steroids influence neurophysin II distribution in the forebrain of normal and mutant mice

The distribution of arginine vasopressin-associated neurophysin (neurophysin II) immunoreactivity was investigated in normal and mutant house mice during development and after various gonadal steroid manipulations. During postnatal development of normal mice dense networks of neurophysin II immunoreactivity in the lateral septal nucleus and lateral habenular nucleus appeared earlier in male than in female mice, with an adult pattern of immunoreactivity being attained by 8 weeks and 12 weeks of age, respectively. The neurophysin II immunoreactivity in the male was denser than that in female mice. After gonadectomy of adult normal mice there was a gradual loss of neurophysin II immunoreactivity in the lateral septum and lateral habenula over a period of 15 weeks. In hypogonadal mice, a mutant in which gonadal development is arrested postnatally due to a deficiency in hypothalamic gonadotrophin releasing hormone, no immunoreactive neurophysin II could be detected in the lateral septum or lateral habenula. A pattern of neurophysin II immunoreactivity similar to that in normal control mice was observed in hypogonadal mice which had been implanted for 4 weeks with silicone elastomer capsules containing testosterone or oestradiol-17 beta, but not 5 alpha-dihydrotestosterone or progesterone. Stimulation of gonadal development and endogenous steroid production in hypogonadal mice by third ventricular grafts of preoptic area tissue from normal neonatal animals also produced a normal pattern of neurophysin II immunoreactivity in the lateral septum and lateral habenula. In the androgen-insensitive testicular feminized mouse immunoreactive neurophysin II was undetectable in the lateral septum and lateral habenula. Treatment of testicular feminized mice with oestradiol-17 beta, but not progesterone, produced a normal pattern of neurophysin II immunoreactivity. The main immunohistological findings were confirmed by radioimmunoassay of tissue extracts which showed that the concentration of arginine vasopressin in lateral septum was far greater in normal males than females and was undetectable in hypogonadal mice; no oxytocin could be detected in the septum of normal or hypogonadal mice. These results show that the expression of neurophysin II immunoreactivity in the lateral septum and lateral habenula of the mouse brain is dependent on the presence of aromatizeable androgens or oestrogens.

Characterization of corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus of Crh-IRES-Cre mutant mice.

Corticotropin-releasing hormone (CRH)-containing neurons in the paraventricular nucleus of the hypothalamus (PVN) initiate and control neuroendocrine responses to psychogenic and physical stress. Investigations into the physiology of CRH neurons, however, have been hampered by the lack of tools for adequately targeting or visualizing this cell population. Here we characterize CRH neurons in the PVN of mice that express tdTomato fluorophore, generated by crosses of recently developed Crh-IRES-Cre driver and Ai14 Cre-reporter mouse strains. tdTomato containing PVN neurons in Crh-IRES-Cre;Ai14 mice are readily visualized without secondary-detection methods. These neurons are predominantly neuroendocrine and abundantly express CRH protein, but not other PVN phenotypic neuropeptides. After an acute stress, a large majority of tdTomato cells express neuronal activation marker c-Fos. Finally, tdTomato PVN neurons exhibit homogenous intrinsic biophysical and synaptic properties, and can be optogenetically manipulated by viral Cre-driven expression of channelrhodopsin. These observations highlight basic cell-type characteristics of CRH neurons in a mutant mouse, providing validation for its future use in probing neurophysiology of endocrine stress responses.

Catecholaminergic Control of Mitogen-Activated Protein Kinase Signaling in Paraventricular Neuroendocrine Neurons In Vivo and In Vitro: A Proposed Role during Glycemic Challenges

Paraventricular hypothalamic (PVH) corticotropin-releasing hormone (CRH) neuroendocrine neurons mount neurosecretory and transcriptional responses to glycemic challenges [intravenous 2-deoxyglucose (2-DG) or insulin]. Although these responses require signals from intact afferents originating from hindbrain CA (catecholaminergic) neurons, the identity of these signals and the mechanisms by which they are transduced by PVH neurons during glycemic challenge remain unclear. Here, we tested whether the prototypical catecholamine, norepinephrine (NE), can reproduce PVH neuroendocrine responses to glycemic challenge. Because these responses include phosphorylation of p44/42 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases 1/2 (ERK1/2)], we also determined whether NE activates ERK1/2 in PVH neurons and, if so, by what mechanism. We show that systemic insulin and 2-DG, and PVH-targeted NE microinjections, rapidly elevated PVH phospho-ERK1/2 levels. NE increased Crh and c-fos expression, together with circulating ACTH/corticosterone. However, because injections also increased c-Fos mRNA in other brain regions, we used hypothalamic slices maintained in vitro to clarify whether NE activates PVH neurons without contribution of inputs from distal regions. In slices, bath-applied NE triggered robust phospho-ERK1/2 immunoreactivity in PVH (including CRH) neurons, which attenuated markedly in the presence of the α1 adrenoceptor antagonist, prazosin, or the MAP kinase kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Therefore, at a systems level, local PVH delivery of NE is sufficient to account for hindbrain activation of CRH neuroendocrine neurons during glycemic challenge. At a cellular level, these data provide the first demonstration that MAP kinase signaling cascades (MEK→ERK) are intracellular transducers of noradrenergic signals in CRH neurons, and implicate this transduction mechanism as an important component of central neuroendocrine responses during glycemic challenge.

Mediation of dehydration-induced peptidergic gene expression in the rat lateral hypothalamic area by forebrain afferent projections

We have previously shown in dehydrated rats that cellular levels of the mRNAs encoding the precursor peptides for corticotropin‐releasing hormone and neurotensin/neuromedin N significantly increase in a restricted region of the lateral hypothalamic area (Watts, 1992, Brain Res. 581:208–216). The experiments reported here address the role that forebrain osmosensitive cell groups or regions associated with autonomic regulation play in developing this mRNA response. The first experiment showed that unilateral knife cuts placed between the rostral forebrain and the lateral hypothalamic area (LHA) will unilaterally attenuate the mRNA response in the LHA to dehydration. In a second experiment, small injections of the retrograde tracer Fluorogold into the region of the LHA containing these mRNAs revealed a direct input from the osmosensitive median preoptic nucleus and subfornical organ and from the fusiform nucleus of the bed nuclei of the stria terminalis, which is part of a complex of cell groups associated with autonomic regulation. We found that at least 30% of the neurons in the median preoptic nucleus and subfornical organ and 14% of the neurons in the fusiform nucleus of the bed nuclei of the stria terminalis that project to the LHA responded to a rapid increase in plasma osmolality with increased c‐fos mRNA levels. In the final experiment, injections of Fluorogold into the LHA were made simultaneously with ipsilateral rostral knife cuts. Here the numbers of neurons accumulating Fluorogold in the median preoptic nucleus, subfornical organ, and the fusiform nucleus were all significantly decreased concomitantly with attenuated mRNA responses in the LHA to dehydration. We conclude that the LHA receives direct and functional projections from the median preoptic nucleus, subfornical organ, and the fusiform nucleus. These projections appear capable of mediating a substantial part of the response of peptidergic mRNAs in the LHA to dehydration.