Cross-modal innervation of primary visual cortex by auditory fibers in congenitally anophthalmic mice.

Auditory-visual cross-modal innervation was examined in control (sighted, ZRDCT-N) and congenitally anophthalmic (eyeless, ZRDCT-AN) mice using electrophysiological recording and pathway tracing with carbocyanine dyes. Electrophysiological data demonstrate that the primary visual cortex of congenitally eyeless, blind, mice receives auditory stimuli. Neuroanatomical data demonstrate a direct connection between the inferior colliculus (IC) and visual cortex. Our experiments provide new information about how the brain adapts to the loss of sight.

Physiological and anatomical properties of the suprachiasmatic nucleus of an anophthalmic mouse.

Congenitally anophthalmic mice (ZRDCT-AN) have circadian rhythms which 'free-run' and are not light modulated. Their rhythms differ from those of controls in: duration of circadian period, length of active phase, and pattern/intensity of activity. Three different populations have been described based upon wheel-running: rhythmic with stable period, rhythmic with unstable period and arrhythmic. Circadian rhythms are generated by neurons in the suprachiasmatic nuclei (SCN) of the hypothalamus. To better understand whether intrinsic properties of SCN neurons differ in anophthalmic and sighted mice, we examined the electrical activity of these neurons in slices, using single unit recordings, ionophoresis and bath perfusion of agonists and antagonists of known SCN neurotransmitters. Lucifer yellow was injected to characterize morphology. In controls, in daytime, units fired at a higher rate (44% at >/=5 Hz) than at night (21% at >/=5 Hz) and with regular interspike intervals versus irregular intervals nocturnally. In anophthalmics four firing patterns were observed as follows: (1) irregular at <5 spikes/s (70% of the total); (2) regular at >/=5 spikes/s; (<10%); (3) irregular bursts (20%); (4) regular bursts (<1%). Most neurons were inhibited by GABA, but a few were excited in controls. Blocking synaptic transmission with low Ca(2+)/high Mg(2+) increased the frequency and regularized the pattern of previously irregular discharges both in anophthalmics and controls. Bicuculline (10(-5) M), a GABA(A) antagonist, had a similar effect. These data suggest that the characteristic irregular firing pattern of anophthalmics, and of controls at night, results from extrinsic, at least in part, GABAergic input.

The relationship between circadian rhythmicity and vasoactive intestinal polypeptide in the suprachiasmatic nucleus of congenitally anophthalmic mice.

To date, the search for the clock component that is both necessary and sufficient for generation of circadian rhythms has relied primarily on experimental interventions such as lesions and transplantation of fetal SCN. While these approaches have been fruitful, lesions disrupt adjacent host tissue and fiber pathways, and donor tissue is likewise subject to trauma during harvest and transplantation. The current investigation has used congenitally anophthalmic (eyeless) mice to ask whether VIP-IR SCN neurons are necessary and sufficient for generation of circadian rhythms. In this animal model, arrhythmic mice occur naturally, together with their rhythmic littermates. We have combined recording of wheel-running activity with light microscopic immunocytochemistry for vasoactive intestinal polypeptide (VIP) and cytoarchitectural analysis of the suprachiasmatic nuclei (SCN) in rhythmic and arrhythmic anophthalmic mice. Our data provide the first definitive evidence that the presence of VIP neurons in the SCN is not sufficient for generation of circadian locomotor rhythms.

Nonphotic entrainment of activity and temperature rhythms in anophthalmic mice.

Although it is more common to study the effects of light on circadian systems, nonphotic stimuli can also influence and entrain circadian clocks. Because anophthalmic mice (ZRDCT-AN) have a genetic mutation that prevents the development of the eyes, they do not respond to light or entrain to light-dark cycles. Thus, entrainment of anophthalmic mice requires a nonphotic zeitgeber (entraining stimulus). In the current study we attempted to entrain sighted and anophthalmic mice of the same strain, using restricted access to an unlocked running wheel as the zeitgeber. First, free-running rhythms were established. The running wheels were then locked, and unlocked only from 0930-1130 h each day. Finally, a postentrainment free run was measured. In one group of animals, body temperature and general activity were measured using a Minimitter telemetry system. In another, general activity was measured by a sensitive force plate beneath the cage. Running-wheel activity was recorded in both groups. The force plate proved satisfactory for observing the behavior of the circadian system during wheel locking, and preferable to the temperature transmitters for long-term studies because the battery life of the mouse temperature transmitters was limited. Both sighted and anophthalmic mice were able to entrain to restricted wheel access, although not all animals responded. Mice that did not entrain showed either no effect of wheel locking or exhibited masking.

Apoptosis in early ocular morphogenesis in the mouse.

Development of the eye requires complex interactions between tissues, extracellular matrix and growth factors. Most cells of the optic primordia grow and differentiate into discrete ocular structures; however, other cells have death as their developmental fate. The most common mechanisms of cell death are apoptosis and necrosis. We have identified the cell death that occurs during ocular morphogenesis in ZRDCT-N mice as apoptosis. Mouse embryos, ages E8.5-E11.5, were embedded in paraffin, sectioned at 5 microns and stained with hematoxylin or by the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The spatial and temporal distribution of apoptotic cells was mapped at 0.5 day intervals using a computerized image analysis system, and 3-D reconstructions were made at each embryonic age. Our data indicate that apoptosis plays a role in normal ocular morphogenesis and provides the groundwork for studies of abnormal ocular development.

Daily patterns of running wheel activity in male anophthalmic mice.

Circadian rhythms are generated by the suprachiasmatic nuclei (SCN) and synchronized (entrained) to environmental light-dark cycles by the retinohypothalamic tract (RHT), a direct pathway from the retina to the suprachiasmatic nuclei. In anophthalmic mice, the optic primordia are resorbed between embryonic days 11.5 and 13, before retinal ganglion cells emerge. Thus the retinohypothalamic tract, which is the primary "zeitgeber" for circadian rhythms in sighted animals, never forms, and there is no retinal or photic input to the circadian system. We have used wheel running activity, a highly consistent and reliable measure of circadian rhythmicity in rodents, to establish the properties of endogenous locomotor rhythms of anophthalmic mice. We have identified three subpopulations of anophthalmic mice: a) rhythmic with strong stable circadian period but significantly increased period length; b) rhythmic with unstable circadian period; and c) arrhythmic. Future correlation of locomotor rhythms with properties of the suprachiasmatic nuclei in these mice will clarify the relationship between generation and properties of circadian rhythms and the neuroanatomical, neurochemical, and molecular organization of the circadian clock.

Diurnal variations in vasoactive intestinal polypeptide-like immunoreactivity in the suprachiasmatic nucleus of congenitally anophthalmic mice.

The present study has combined recording of circadian locomotor rhythms with light microscopic immunocytochemistry for vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN) of congenitally anophthalmic mice. These mice, which never develop retinae or optic nerves and do not perceive light, are thus in constant darkness. Our data show a circadian rhythm in expression of VIP in the SCN of anophthalmic mice--expression is maximal during late subjective night/early subjective day and minimal in late subjective day/early subjective night. These observations support the hypothesis that expression of VIP is related to regulation of circadian rhythms by the SCN.

The geniculohypothalamic pathway in a congenitally anophthalmic mouse.

In a previous study we described abnormalities in cytoarchitecture and vasoactive intestinal polypeptide distribution in the suprachiasmatic nucleus (SCN) of anophthalmic mice. However, the effect of anophthalmia on the geniculohypothalamic pathway, an important pathway for relay of photic information to the SCN, is not known. The present study examined the geniculohypothalamic pathway in congenitally anophthalmic and sighted control mice. The data demonstrate that the development of an intergeniculate leaflet (IGL), the expression of neuropeptide Y (NPY) by IGL neurons and the formation of NPY terminal fields in the SCN proceed in the absence of retinal input. Although the cytoarchitectural organization of the anophthalmic IGL differs from that of the control mouse, the distribution of NPY plexuses in the suprachiasmatic nucleus is remarkably similar.

VIP-like immunoreactivity in the suprachiasmatic nuclei of a mutant anophthalmic mouse.

The suprachiasmatic nuclei (SCN) of mutant anophthalmic (strain ZRDCT-An) and sighted control mice was examined using light microscopic methods for cytoarchitecture and immunocytochemistry for vasoactive intestinal polypeptide (VIP). The SCN of the anophthalmic mice were asymmetrical, and quite variable in cytoarchitecture. Immunocytochemistry for VIP revealed strong staining of cells and fibers. Within the SCN, the distribution of VIP-immunoreactive cells was more diffuse in anophthalmic mice than in the controls; immunoreactive neurons were frequently observed in ectopic locations in the mutant animals.

Neuropeptide Y-like immunoreactivity in the diencephalon of the little brown bat (Myotis lucifugus): localized variations with physiological state.

The current study used light microscopic immunocytochemistry to demonstrate and compare neuropeptide Y-like immunoreactivity (NPY-IR) in the diencephalon of the little brown bat (Myotis lucifugus) at different stages in its annual cycle of activity and hibernation. Animals were sacrificed in each of three discrete physiological states: euthermic, hypothermic, and hibernating. In general, NPY-IR was abundant in the hypothalamus and sparse in other diencephalic areas. Immunoreactivity was present in a number of pathways which project to or originate from diencephalic nuclei; these include the ansa peduncularis, medial forebrain bundle, inferior thalamic peduncle, stria terminalis, stria medullaris, mammillary peduncle, and dorsal longitudinal fasiculus. Dense fiber plexuses were present throughout the hypothalamus; however, NPY-IR was conspicuously absent from the suprachiasmatic nucleus. Immunoreactive perikarya were located in the supraoptic, dorsomedial, ventromedial, and arcuate nuclei, in the external division of the ventral lateral geniculate nucleus, and in the pineal gland. Localized changes in density and/or distribution of NPY-IR were correlated with changes in physiological state.

Unilateral enucleation alters vasoactive intestinal polypeptide-like immunoreactivity in the suprachiasmatic nucleus of the rat.

Adult Sprague-Dawley rats were unilaterally enucleated under deep anesthesia and sacrificed 3-11 months post-operatively. The brains were removed and the suprachiasmatic nuclei were evaluated for vasoactive intestinal polypeptide (VIP)-like immunoreactivity. Our data show that expression of VIP is reduced in the nucleus that is deprived of retinal input.

Synaptogenesis in the rat suprachiasmatic nucleus: a light microscopic immunocytochemical survey.

MabQ155, a monoclonal antibody against synaptophysin, has been used to conduct a light microscopic survey of synaptogenesis in the suprachiasmatic nucleus of the perinatal rat. Synaptophysin is an integral component of synaptic vesicle membranes which is expressed in growth cones and growth cone filopodia as well as in mature synapses. With the light microscope, mabQ155 immunoreactivity in growth cones can be distinguished from that in presynaptic terminals on the basis of the size of immunoreactive puncta. The current study presents a qualitative and quantitative analysis of synaptogenesis from the day of birth (P0) to postnatal day 10 (P10). In our quantitative analysis we have used daily intervals during the first postnatal week, distinguished between growth cones and presynaptic terminals, and divided the suprachiasmatic nucleus into sampling regions that are related to the progress of synaptogenesis. Our data demonstrate regional differences in synaptogenesis within the suprachiasmatic nucleus (SCN), document the temporal progression from the penetration of growth cones to the appearance of mature synapses, and provide information about gradients of synaptogenesis in the nucleus during development.

Neuropeptide Y innervation of the cerebellum in the little brown bat (Myotis lucifugus).

Light microscopic immunocytochemistry for neuropeptide Y (NPY) has demonstrated the presence of a diffuse system of NPY-immunoreactive fibers in the cerebellum of Myotis lucifugus. These fibers gain access to the cerebellum by way of the superior cerebellar peduncle, and terminate on Purkinje cell dendrites and on neurons in the granule cell layer. The origin of cerebellar NPY and possible colocalization with norepinephrine are discussed.

The maturation of the somatostatin systems in the rat visual cortex.

The development of somatostatin-immunoreactive (SS) neurons and processes in the rat visual cortex (VC) was studied in animals from embryonic day 20 (E20) to postnatal day 21 (D21). Three distinct patterns of immunoreactivity were seen. From E20 to birth (D0), VC was characterized by a small number of mainly bipolar SS neurons throughout the cortical plate. In the perinatal period, from D1 to D6, there were large numbers of immature immunoreactive neurons which were confined to layer VI and the subplate zone, a few bipolar neurons in the cortical plate and an extremely dense plexus of SS processes throughout the neuropil. The third phase, from D8 to weaning, was characterized by the absence of immature SS neurons, an increase in the number of multipolar SS neurons and a decrease in the density of SS fibers. By D15, the time of eye-opening, the number and distribution of SS neurons and processes was close to that seen in the adult. These results indicate that the SS system of neurons and fibers is among the earliest of the transmitter systems to be established in VC and suggests a role for the peptide in cortical organization as well as visual processing.

Cholecystokinin (CCK)-like immunoreactivity in the brain of the little brown bat (Myotis lucifugus).

The distribution of Cholecystokinin (CCK-8)-like immunoreactivity was mapped in the brain of the little brown bat, Myotis lucifugus, at three different and discrete levels of physiological activity: euthermic, hypothermic, and hibernating. Immunoreactive perikarya were present in the cerebral cortex, hippocampal formation, several nuclei of the olfactory and limbic systems, the ventral lateral geniculate nucleus, suprachiasmatic nucleus, medial geniculate nucleus, and caudate-putamen. Immunoreactive fibers were present in plexuses throughout the brain and in three major projection pathways: the medial forebrain bundle, the mammillary peduncle, and dorsal longitudinal fasciculus. Our data suggest two possible loci for CCK regulation of feeding behavior: a hypothalamic locus in the dorsomedial nucleus and a brainstem locus in the nucleus tractus solitarius.

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the suprachiasmatic nucleus of the perinatal rat.

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was examined in the suprachiasmatic nucleus (SCN) of the perinatal rat using the unlabeled antibody enzyme method of Sternberger. Our results showed that VIP-like immunoreactivity was present in cells and fibers of the SCN prior to birth. Immunoreactive neurons at this time appeared morphologically immature, and their distribution differed somewhat from that seen in the adult SCN.

Immunocytochemical localization of vasoactive intestinal polypeptide (VIP) in the brain of the little brown bat (Myotis lucifugus).

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity has been examined in the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry and the indirect antibody enzyme method of Sternberger. Animals were sacrificed at three different and discrete levels of physiological activity: euthermic, hypothermic and hibernating. The density and distribution of immunoreactive neurons and fibres was compared in the three animal groups with the aid of a computerized image analysis system. Our results were compared with those of previous studies in laboratory species such as the rat and cat. Our study has demonstrated marked changes in the density of VIP-immunoreactive fibres and plexuses in the anterior hypothalamic area which correspond to the physiological state of the animal. In addition we have demonstrated the presence of VIP immunoreactive perikarya in a number of previously unreported locations. These include the paraventricular and periventricular hypothalamic nuclei, the linear raphe nucleus, nucleus interfascicularis, and in neurons embedded in the fibres of the dorsal tegmental decussation.

VIP-like immunoreactivity in the anterior hypothalamic area of a hibernating bat varies with physiological state.

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the anterior hypothalamic area (AHA) was studied in 15 bats which are seasonal hibernators, using the unlabeled antibody enzyme method of Sternberger. Our results showed that the density of immunoreactive fibers and terminal plexuses in the AHA was greatest in euthermic animals and decreased dramatically when body temperature and cardiac rate were depressed and animals entered hibernation.

Distribution of somatostatinlike immunoreactivity in the brain of the little brown bat (Myotis lucifugus).

The distribution of somatostatinlike immunoreactive (SLI) perikarya, axons, and terminals was mapped in subcortical areas of the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry. A preponderance of immunoreactivity was localized in reticular, limbic, and hypothalamic areas including: 1) in the forebrain: the bed nucleus of the stria terminalis; lateral preoptic, dorsal, anterior, lateral and posterior hypothalamic areas; amygdaloid, periventricular, arcuate, supraoptic, suprachiasmatic, ventromedial, dorsomedial, paraventricular, lateral and medial mammillary, and lateral septal nuclei; the nucleus of the diagonal band of Broca and nucleus accumbens septi; 2) in the midbrain: the periaqueductal gray, interpeduncular, dorsal and ventral tegmental, pretectal, and Edinger-Westphal nuclei; and 3) in the hindbrain: the superior central and parabrachial nuclei, nucleus incertus, locus coeruleus, and nucleus reticularis gigantocellularis. Other areas containing SLI included the striatum (caudate nucleus and putamen), zona incerta, infundibulum, supramammillary and premammillary nuclei, medial and dorsal lateral geniculate nuclei, entopeduncular nucleus, lateral habenular nucleus, central medial thalamic nucleus, central tegmental field, linear and dorsal raphe nuclei, nucleus of Darkschewitsch, superior and inferior colliculi, nucleus ruber, substantia nigra, mesencephalic nucleus of V, inferior olivary nucleus, inferior central nucleus, nucleus prepositus, and deep cerebellar nuclei. While these results were similar in some respects to those previously reported in rodents, they also provided interesting contrasts.

Bilateral projections of neurons in the lateral geniculate nucleus and nucleus lateralis posterior to the visual cortex in the neonatal rat.

The projection from the lateral geniculate nucleus (LGN) and nucleus lateralis posterior (LP) to the visual cortex was examined in rat pups 3-7 days of age using the fluorescent tracers True Blue, Fast Blue and Nuclear Yellow. Our data provide the first evidence that (1) these projections are bilateral, (2) the ipsilateral projection from these nuclei to the visual cortex in the neonatal rat is well localized and is similar in distribution and organization to that reported by others in the adult and (3) bilaterally projecting geniculocortical cells are morphologically heterogeneous; bilaterally projecting cells in LP are morphologically homogeneous.