Neuronal changes in the forebrain of mice following penetration by regenerating olfactory axons.

Following total, unilateral bulbectomy in neonatal mice, the olfactory sensory axons regrow from a reconstituted population of sensory neurons, cross the lamina cribrosa, and invade the spared forebrain that has leaned forward toward the anteroventral wall of the cranial cavity. The sensory axons invade several regions of the spared forebrain, at times penetrating deeply into the brain parenchyma. These axons terminate in characteristic globose structures resembling the glomeruli of the olfactory bulb. However, they can be distinguished from the latter by the absence of periglomerular cells. These ectopic glomerular structures are formed by the commingling of the olfactory axon terminals and the dendrites of brain neurons that lie in their proximity. Previously we have established that synaptic contacts occur between the sensory axon terminals and the dendrites of the brain neurons. Our present study describes large neurons, resembling mitral cells, that expand their dendrites into the intracerebral glomeruli. These neurons are recognized by virtue of their relatively large diameter, their selective stainability with silver methods, and the unorthodox arrangement of their dendrites in comparison with the neurons of the region. Their appearance is contingent upon the presence of ectopic glomeruli. The possibility is discussed that the large argyrophilic neurons may be derived from developing neuronal elements of the brain.

Neonatally bulbectomized rats with new olfactory-neocortical connections are anosmic.

Rats with one olfactory bulb removed when neonates and the second bulb removed when adults were tested on tone-light discrimination and odor detection tasks. On the neonatally operated side reconstituted olfactory receptor cell axons penetrated the frontal neocortex or portions of the anterior olfactory nucleus, and formed glomerular-like structures. On the adult operated side there was extensive scar formation which prevented in-growing sensory axons from contacting the brain. All experimental animals acquired the tone-light discrimination but failed to show any evidence of odor detection. These results indicate that reconstituted olfactory projections which terminate in the frontal neocortex or anterior olfactory nucleus do not support olfaction.

Experimental studies on the olfactory marker protein. III. The olfactory marker protein in the olfactory neuroepithelium lacking connections with the forebrain.

Total unilateral bulbectomy induces degeneration of the mature olfactory neurons and disappearance of the olfactory marker protein from the primary sensory pathway. Owing to the presence of a neurogenetic matrix in the neuroepithelium, reconstitution of a new population of neuronal elements occurs. In this experiment, connections of the regrown olfactory axons with the spared forebrain are barred by the formation of scar tissue. In spite of the absence of a target, new neurons differentiate and produce olfactory marker protein.

Experimental studies on the olfactory marker protein. II. Appearance of the olfactory marker protein during differentiation of the olfactory sensory neurons of mouse: an immunohistochemical and autoradiographic study.

The time interval between the incorporation of [3H]thymidine and the appearance of olfactory marker protein (OMP) in autoradiographically labeled neurons which have differentiated from stem cells, has been determined by autoradiographic and immunohistochemical techniques. The first [3H]thymidine-labeled, OMP-containing elements have been observed 7 days after administration of the radioactive thymidine. This result allows some speculation on the potential function of the olfactory marker protein.

Experimental studies on the olfactory marker protein. I. Presence of the olfactory marker protein in tufted and mitral cells.

Partial, unilateral olfactory nerve section was performed in mice, and the behavior of the olfactory marker protein (OMP) studied, after this experimental manipulation, with the peroxidase--antiperoxidase method. The protein, which in normal mice is present only in mature olfactory sensory neurons, after unilateral lesion of the fila olfactoria was observed in mitral and tufted cells of both olfactory bulbs. Positive elements, present at 5 days postoperative, increased in number up to 30 days and some could still be detected at 60 days. The functional implications of this finding are briefly discussed.

Reinnervation of the olfactory bulb after section of the olfactory nerve in monkey (Saimiri sciureus).

Section of the fila olfactoria in squirrel monkey, a non-human primate, induces rapid degeneration of the sensory axon terminals in the olfactory bulb glomeruli. A population of axons, from newly formed sensory neurons in the olfactory neuroepithelium, regrow, passes the lamina cribrosa and, upon reaching the olfactory bulb, reinnervates the glomeruli. A new set of synaptic contacts is reformed between the sensory terminals and the post-synaptic dendritic processes of the glomeruli. Our observations indicate that this portion of the CNS of a non-human primate can be reinnervated after deafferentiation, and that active synaptogenesis occurs.

Neurogenesis and neuron regeneration in the olfactory system of mammals. III. Deafferentation and reinnervation of the olfactory bulb following section of the fila olfactoria in rat.

Axotomy at the level of the lamina cribrosa in rat induces rapid degeneration of the olfactory sensory axons in the bulb. The phenomenon, which is limited to the layer of olfactory fibres and to the glomeruli of the bulb, can be observed as early as 15-24 h after surgery, and peaks at 3-4 days. The glomeruli located in the rostro-ventral portion of the bulb are affected first, and the process extends to the dorso-caudal portion with a delay of 12-24 h. Reactive hypertrophy of the glia coincides with removal of the degenerating terminals, and is maximal 48 h after axotomy. Axotomy does not preclude reinnervation of the bulb by axons originating from new, reconstituted neurons in the olfactory neuroepithelium. These new axons begin to reach the periphery of the bulk approximately at the 20th day post-operative and then reinnervate the glomeruli. The rostro-ventral portion of the bulb is the first to be reinvaded by the new axons. The glomeruli reacquire a morphological pattern similar to controls between 20 to 30 days.

Immunocytochemistry of the olfactory marker protein.

The olfactory marker protein has been localized, by means of immunohistochemical techniques in the primary olfactory neurons of mice. The olfactory marker protein is not present in the staminal cells of the olfactory neuroepithelium, and the protein may be regarded as indicative of the functional stage of the neurons. Our data indicate that the olfactory marker protein is present in the synaptic terminals of the olfactory neurons at the level of the olfactory bulb glomeruli. The postsynaptic profiles of both mitral and periglomerular cells are negative.

Denervation in the primary olfactory pathway of mice. IV. Biochemical and morphological evidence for neuronal replacement following nerve section.

Unilateral olfactory nerve section was performed in the mouse. Three biochemical markers of the olfactory chemoreceptor neurons: carnosine, carnosine synthetase activity and the olfactory marker protein, were measured in the olfactory bulb and epithelium. Parallel observations were made by light microscopy as well as at the ultrastructural level. The specific biochemical markers decrease rapidly in both bulb and epithelium and reach a minimum by the end of the first week after surgery. They then slowly return to 80% of control values by one month. Carnosinase activity in epithelium was essentially unaffected. These biochemical observations coincide temporally with the onset of degenerative changes seen morphologically, in both the bulb and epithelium. The degenerative changes persist for up to two weeks in the bulb and for about one week in the epithelium. At this time basal cell division and differentiation begins in the epithelium with subsequent regrowth of olfactory axons into the glomerular layer of the olfactory bulb with ther reappearance of olfactory axon terminals. The temporal coincidence of these biochemical and morphological observations suggests they are manifestations of the same process, and is consistent with the idea that the olfactory chemoreceptor neurons are perhaps unique in being able to be replaced from undifferentiated stem cells.

Olfactory epithelium of Necturus maculosus and Ambystoma tigrinum.

The morphological study presented here provides a general description of the elements of the olfactory epithelium in the mud puppy and tiger salamander,, and gives evidence about their dynamic activity and interrelationships. There are morphological indications of local bursts of reduplication and a continual line of differentiation of receptor cells from basal cell progenitors through stages of mature development to senescence (indicated by the accumulation of pigment granules) and cell death and disposal (by expulsion of pycnotic cell nuclei and by phagocytosis by macrophages). The supporting cells probably play several roles: a secretory role which supplements the activity of Bowman's glands, a minor insulating role in which some dendrites are shielded from the surrounding milieu, and a skeletal role in which they facilitate the efficient displacement of dendrites. The dendrites are regularly arranged in organized relationships with one another and are for the most part in direct apposition, separated only by a 200 A intercellular gap, thus suggesting the possibility of functional interrelationships. This study emphasizes the fact that efficient planning of experimental investigations must include knowledge and consideration of the thickness of the particular olfactory epithelium under study. It also suggests that because of the large receptor-cell size, the mud puppy and/or tiger salamander would make good model systems for single cell recording. Further, the olfactory epithelia of these species are suggested as favorable targets for studies of the aging process in nerve cells.